Evaluation of Alnus nitida (Spach) Endl. for Pharmacological Activities

Abstract

The aimmof present studyowas to screen pharmacological potential of Alnus nitida leaves, bark, fruit and their deriveddfractions; n-hexane,chloroform, ethyl acetateiand the soluble residualkaqueous fraction. Qualitative screening of methanol extracts demonstrated the occurrence of terpenoids, anthraquinones, coumarins, cardiac glycosides, saponins, terpenoids, coumarins, phenols, betacyanin, flavonoids, tannins, phlobatannins and alkaloids. HPLC analysis revealed theypresence of standards; catechin, gallic acid, rutin, quercetin, myricetin andycaffeic acid in ANLM, ANBM, ANFM and their various fractions in varying concentrations. GCMS chemical fingerprinting of ANLM, ANBM and ANFM depicted the presence of 30 different chemical constituents belonging to diverse classes, owing enormous biological activities. Methanol extract, ethyl acetate and aqueous fractions demonstrated the high level of total flavonoid and phenolic contents and a strong correlation coefficient with the IC50 values were determined for the quenching of DPPH, hydroxal radical, nitric oxide radical, ironnchelating,iβ-carotenebleaching inhibition,itotal antioxidant activity and for total reducing power assay. Significant antimicrobial and cytotoxic action was demonstratediby methanoliextract and ethyl acetateifractions of leaves, bark and fruit. Methanoluextract, ethyl acetateyand aqueoushfractions of leaves, bark and fruit also revealed marked anti-inflammatory and anti-diabetic potential. The in vivo study indicated that A. nitida leaves, bark and fruit possessed potent antioxidant activity against CCl4 induced toxicity in rats. Treatment of rats with ANLM, ANBM and ANFM markedly improved the serum biomarkers of organ toxicity and tissue antioxidant status by significantly ameliorating the oxidative tissues markers enzymes levels near to control. Histopathological studies of different organs verify the biochemical observations. Comet assay was done to assess the defensive potential of ANLM, ANBM and ANFM treated organs against CCl4 stimulated DNA damage. Various in vivo anti-inflammatory assays were done by carrageenan inducedgpaw edema,gFreund’s completetadjuvant arthritis, histamine induced paw edema and xylene induced ear edema in rats. Analgesicoactivity was assessed by hotiplate analgesic test and aceticfacid inducediwrithing test. Chloroform fraction of ANL, ANB and ANF was best active as anti-inflammatory and analgesic. In vivo anti-diabetic activity revealed ethyl acetate and aqueous fractions of ANL and ANF as significant anti-diabetic and antioxidant agent in multiple organs against xv alloxan induced toxicity. Histological evaluation and comet assay also illustrated the protective ability of ethyl acetate and aqueous fractions of ANL and ANF. In vitro models of lung cancer cells (A-549 and H460) were usedito evaluate the anticancer potentiallof crude extracts (ANL, ANB) and their isolatedcompounds (RU, MI). ANL, ANB, RU and MI inhibit cell viability and proliferation in A-549 and H460 cells in a dose dependent manner. Extracts/compounds induce cell death via suppressing various signal transduction pathways that regulates cell proliferation and survival. Chromatinkcondensation, cell shrinkageiand apoptoticibodies were observed by phase contrast microscopy. Extracts/compounds significantly inhibitedicell survivaloand colonyigrowth in bothicell lines. Migration studies was also done by wound scratch and transwell assay, ANL, ANB, RU and MI significantly inhibited migration rate in a doseidependent manner. Staining with DAPI and phalloidin Factin staining demonstratedhthat cell deathioccurred at least partly through induction of apoptosis9in both cell lines. Cell cycle analysis was also done which resulted in arrest of cell at G1 phase, which might occur due to modulation of cyclin D1 expression, detected by western blot. ANL, ANB, RU and MI repressed the expression ofuanti-apoptotic proteins Bcl-2, xIAP and Bcl-xL in dose dependent manner, which futher validate the apoptotic effect of extracts and isolated compounds. Further analysis of signaling pathways indicated that compounds treatment induced a dose dependent suppression of PI3-K, p-Akt (Ser473and Thr308), NFκB p65, p- ERK1/2 (Thr202/Tyr204) in A-549 and H460 cells. In vivo lung metastasis studies depicted that ANL and ANB have anti-metastatic property. The anti-metastatic function of ANL and ANB is supportedkby the fact that iticould inhibitythe formationiof nodules on the lungjtissue in C57BL/6J mouse lung metastaticcmodel usinguB16F10 melanomaicells. Western analysis illustrated that ANL and ANB decreased phosphorylation of FAK protein expression innvivo. FAK is a validjtherapeuticitarget againstumelanoma. Results also depicted that these extracts have anti-metastaticcproperties possiblyivia itsianti-angiogenesisiinduced byydownregulation9of VEGF. ANL and ANB also decreased the expression of anti-apoptotic protein Bcl-2 and Bcl-xL in vivo. These findings provide strong indication that A. nitida extracts/compounds may be favorable therapeutic candidates against two human non-small cell lung carcinoma cells.