Chemical and Biological Studies of Some Litsea Species Available in Bangladesh

Abstract

In order to discover new phytochemicals as well as searching for evidence based information of traditional therapeutic uses of medicinal plants, four species of the genus Litsea belonging to the Lauraceae family were selected in this study. The selected plants are Litsea glutinosa Lour., L. monopetala Roxb., L. deccanensis Gamble. and L. lancifolia Hook. f. Two compounds were revealed from L. glutinosa and they are 4΄-O-methyl-(2 ̋,4 ̋,-di- E-p-coumaroyl)-afzelin (LGC-26, 95) and quercetin-3-O-(2 ̋,,4 ̋,-di-E-p-coumaroyl)-α- L-rhamnopyranoside [or, 5΄-hydroxyl-(2 ̋,4 ̋-di-E-p-coumaroyl) afzelin] (LGC-45-3, 96). Both of the compounds were reported for the first time from Litsea species. Five compounds were isolated and purified from the leaf extract of L. monopetala and characterized as vomifoliol (LML 363-1, 97), α-amyrin (LML 309, 98), β-amyrin (LML 301, 99), (E)-6,7,8,9,10,11-hexahydro-8,17:10,16-di(metheno)dibenzo- [h,l][1]oxa[5] azacyclotridecine-1,4-diol (LML 339-1, 100) and (Z)-1,2,3,4,5,6- hexahydro-8,11-etheno-2,13:4,12di(metheno)benzo[h][1]-oxa[5]aza-cyclopentadecine (LML 339-2, 101) by 1H and 13C NMR, COSY, HSQC, HMBC spectral data analysis. All these five compounds are reported for the first from L. monopetala, while compounds 101 and 101 appear to be new compounds. The obtained 1H NMR spectral data and the comparison with the reference value helped us to characterize lupeol (LDC-10-3, 102) and a mixture (4:1 ratio) of β-sitosterol and stigmasterol (LDC-10-2, 55 & 56) from L. deccanensis and β-sitosterol (LLC-10-1, 55) from L.lancifolia. Lupeol has been isolated from L. deccanensis for the first time. 4ʹ -O-methyl (2 ̋,4 ̋-di-E-p-coumaroyl) afzelin (95) Quercetin3-O-(2 ̋,4 ̋-di-E-p-coumaroyl)-α-Lrhamnopyranoside [or 5΄-hydroxyl-(2 ̋,4 ̋-di-E-p-coumaroyl) afzelin] (96) The crude extracts of L. glutinosa, L. monopetala, L. deccanensis and L. lancifolia were evaluated for biological activities through in-vitro and in-vivo screenings. For antidiarrheal activity test, 100, 200 and 400 mg/kg bw methanol extract of L. glutinosa (MELG), L. monopetala (MELM), L. deccanensis (MELD) and L. lancifolia (MELL) were administered in two animal models where 0.5 ml castor oil was used for diarrhea induction and 3 mg/kg bw loperamide was used as standard drug. In all the groups treated with MELG, MELM, MELD and MELL extracts wet feces number, total number of feces and total weight of the foecal output were decreased significantly (p<0.05) with rising of doses. The maximum peristaltic inhibition was observed 32.36%, 22.52%, 26.26% and 33.22% for 400 mg/kg by MELG, MELM, MELD and β- amyrin (99) (E)-6,7,8,9,10,11-hexahydro- 8,17:10,16-di(metheno)dibenzo- [h,l][1]oxa[5]azacyclotridecine- 1,4-diol (100) (Z)-1,2,3,4,5,6-hexahydro-8,11-etheno- 2,13:4,12-di(metheno)benzo- [h][1]oxa[5]azacyclopentadecine (101) Stigmasterol (56) Lupeol (102) β-Sitosterol (55) Vomifoliol (97) α- amyrin (98) MELL extracts respectively. The peristaltic indices were 59.0%, 79.0%, 59.1% and 63.0% for 400 mg/kg of MELG, MELM, MELD and MELL respectively compared to the control (90.0%) and standard (66.7%) groups. For all the plant extracts the percentage inhibition of gastrointestinal motility and peristalsis index were comparable to the standard. In analgesic activity test, MELG, MELM, MELD and MELL on acetic acid-induced writhing in mice at two different doses (100 and 200 mg/kg bw) showed significant reduction of squirming (p<0.001, p<0.01 and p<0.05) in a dose dependent manner as paralleled to control. In the second animal model (Eddy’s hot plate method), pain was induced by heat and analgesia was assessed by counting the time required for the initiation of the reaction. Out of the four plants, all the plants at 100, 200 and 400 mg/kg doses, increased the latency time. The pain-relieving activity data (formalin method) are presented as licking and biting time in seconds at early- and late-phases of treatment with plant extracts. In both the early- and late-phase, reaction time for licking and biting hind paw were decreased with the increment of the doses (from 100 mg/kg bw to 200 mg/kg bw) but in the late phase (20-30 min) the reaction time was decreased significantly (p < 0.05) with the increment of doses for all the studied plant extracts as well as standard indomethacin at 10 mg/kg bw. The effects of MELG, MELM, MELD and MELL on blood glucose level in streptozocin (STZ) induced diabetic rats were found to drop the blood glucose level (BGL) significantly (p<0.05) after 7th days of treatment with the plant extracts at 300 and 500 mg/kg/day doses. Percentage inhibition of blood glucose level for MELG, MELM, MELD and MELL were comparable with that of standard metformin and they are 66.69%, 57.06%, 68.16% and 69.33% respectively at 500 mg/kg/day dose as compared to the untreated diabetic control group. Hole cross test was performed to investigate the possible neuropharmacological effects (CNS stimulant or depressant) of MELG, MELM, MELD and MELL and all the extracts at two different doses (300 and 500 mg/kg bw) found to reduce locomotion in the test animals and to decrease the passing number through the hole in between the hole cross chamber by the animals in a dose dependent manner. Three fractions of four different species of Litsea were studied for antimicrobial activity by disc diffusion method. The results of antimicrobial activity test of different fractions showed mild to moderate activity for L. monopetala, very good activity for L. lancifolia, moderate to good activity for L. glutinosa and mild to very good antimicrobial activity for L. deccanensis against the microorganisms selected for this study. The quantities of phenolic compounds were found in ethyl acetate fraction of L. glutinosa (103.04±0.06), followed by ethyl acetate fraction of L. lancifolia (79.94±0.07). Among the plants L. lancifolia and L. glutinosa have shown very good total phenolics compared to L. deccanensis and L. monopetala. In DPPH free radical scavenging activity test, the IC50 for pet-ether, chloroform and ethyl acetate fractions of L. deccanensis were 31.75 μg/ml, 24.62 μg/ml and 31.04 μg/ml, respectively. All the values are comparable with that of ascorbic acid (31.66 μg/ml). For L. lancifolia 63.97 μg/ml pet-ether, 65.91 μg/ml chloroform and 80.46 μg/ml ethyl acetate extractives were required for 50% scavenging of free radicals. The effective concentrations for pet-ether, chloroform and ethyl acetate fractions of L. glutinosa were measured as 25.19 μg/ml, 37.90 μg/ml and 67.41 μg/ml, respectively. The IC50 values were 31.94 μg/ml, 24.91 μg/ml and 31.10 μg/ml for pet-ether, chloroform and ethyl acetate fractions of L. monopetala, respectively. From results, it may be proposed that three different extractives of L. deccanensis, L. lancifolia, L. glutinosa and L. monopetala were able to exhibit the free radical scavenging activity compared to ascorbic acid, a potent antioxidant compound. The docking simulation was conducted against aldose reductase (AKR1B1) protein model with the purified compound 95 and 96 by using Auto Dock Vina software. Compound 95 exposes the higher negative binding affinity (-9.8 kcal/mol) as compared to the compound 96 with binding affinity (-9.4 kcal/mol) for the interaction of the target protein aldose reductase (AKR1B1). Compound 95 exhibited strong connection with eleven hydrophobic bonds, hydrogen bonds and one other bond while compound 96 developed stable interactions by three hydrogen bonds, and eleven hydrophobic bonds. During investigating interaction pattern, binding affinity, and best binding poses of the compounds it can be proposed that both structures might be promising inhibitors against aldose reductase (AKR1B1) protein. Molecular docking analysis of isolated compounds 95 and 96 (−9.4, and −8.9 kcal/mol, respectively) against human pancreatic alpha amylase showed promising docking affinity. Compound 95 formed polar contacts with Tyr-151, Thr-163, Arg-195, Asp-197, His-201, and His-299 residues and compound 96 showed polar contacts with Gln-63, Arg-195, Asp-197, and His-299 residues. These findings suggest that these compounds are promising inhibitors of human pancreatic alpha amylase. The docking results of vomifoliol clearly indicate that it is a better candidate as an analgesic agent. Vomifoliol (97) is a potent binder (-4.9 kcal/mol) to COX-2 than indomethacin (-1.1 kcal/mol) indicating that it is supposed to have better analgesic action.