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Fulltext Evaluation of Antioxidant Activity and Acute Toxicity of Clausena excavata Leaves Extract

Albaayit SF, Abba Y, Abdullah R, Abdullah N

Evid Based Complement Alternat Med, 2014;2014:975450.
PMID: 25610488 DOI: 10.1155/2014/975450

Clausena excavata (Lour.), locally known as "Kemantu hitam," is a common plant in Malaysian folklore medicine. This study evaluated the antioxidant properties of the solvent extracts of C. excavata leaves and determined the acute toxicity of methanolic extract C. excavata (MECE) leaves in Sprague-Dawley rats. Harvested leaves were dried and subjected to solvent extraction using petroleum ether, chloroform, ethyl acetate and methanol in succession. The antioxidant activity of each extract was determined using the ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picryl dihydrazyl (DPPH) radical scavenging activity. The total phenolic content (TPC) and total flavonoids content (TFC) were estimated by Folin-Ciocalteu and ethanolic aluminium chloride method, respectively. The chloroform extract was found to be highest in flavonoid content, while the methanolic extract showed the highest TPC and antioxidant activity. There was no mortality in rats treated with MECE leaves even at a high dose of 5000 mg/kg body weight. However, the MECE leaves produced mild to moderate pathological changes in the liver and kidneys, shown by mild degenerative changes and leucocyte infiltration. The extract did not affect the haematological parameters or relative weights of the liver or kidneys. Overall, the MECE leaves have potent antioxidant activity and are presumed safe to be used orally as health-promoting product at low to moderate doses.

Matched MeSH terms: Clausena
Fulltext Anti-Enterococcus Faecalis, Cytotoxicity, Phytotoxicity, and Anticancer Studies on Clausena excavata Burum. f. (Rutaceae) Leaves

Albaayit SFA, Maharjan R, Abdullah R, Noor MHM

Biomed Res Int, 2021;2021:3123476.
PMID: 33748267 DOI: 10.1155/2021/3123476

BACKGROUND: Clausena excavata Burum. f. has long been applied in ethnomedicine for the treatment of various disorders like rhinitis, headache, cough, wound healing, fever, and detoxification. This study is aimed at investigating the antibacterial activity against Enterococcus faecalis ATCC 49532 using AlamarBlue assay and atomic force microscopy (AFM) as well as the cytotoxicity, anticancer, and phytotoxicity of C. excavata.
METHOD: Bacterial cell viability was performed by using microplate AlamarBlue assay. Atomic force microscopy was used to determine morphological changes in the surface of bacterial cells. Cytotoxicity and phytotoxicity were determined by brine shrimp lethality and Lemna minor bioassay. Caco-2 (colorectal adenocarcinoma) cell line was used for the evaluation of the anticancer effects.
RESULT: Among the fractions tested, ethyl acetate (EA) fraction was found to be active with minimum inhibitory concentration (MIC) of 750 μg/mL against E. faecalis, but other fractions were found to be insensitive to bacterial growth. Microscopically, the EA fraction-treated bacteria showed highly damaged cells with their cytoplasmic content scattered all over. The LC50 value of the EA fraction against brine shrimp was more than 1000 μg/mL showing the nontoxic nature of this fraction. Chloroform (CH), EA, and methanol (MOH) fractions of C. excavata were highly herbicidal at the concentration of 1000 μg/mL. EA inhibited Caco-2 cell line with an IC50 of 20 μg/mL.
CONCLUSIONS: This study is the first to reveal anti-E. faecalis property of EA fraction of C. excavata leaves, natural herbicidal, and anticancer agents thus highlight the potential compound present in its leaf which needs to be isolated and tested against multidrug-resistant E. faecalis.

Matched MeSH terms: Clausena/chemistry*
First Report of Pantoea anthophila Causing Soft Rot Disease in Clausena lansium (Wampee) in China

Zhou JN, Liu SY, Chen YF, Liao LS

Plant Dis, 2015 Mar;99(3):416.
PMID: 30699721 DOI: 10.1094/PDIS-10-14-1025-PDN

Clausena lansium, also known as wampee (Clausena wampi), is a plant species native to China, Vietnam, the Philippines, Malaysia, and Indonesia, where it is widely cultivated, and also grown in India, Sri Lanka, Queensland, Florida, and Hawaii, but less frequently (3). The fruit can be consumed fresh or made into juice, jam, or succade. In summer to fall 2014, a soft rot disease was found in a wampee planting region in Yunan County, Guangdong Province, China. On Sept. 18, we collected diseased samples from a wampee orchard with about 20% disease incidence. The infected fruit initially showed pinpoint spots on the peel, water-soaked lesions, and light to dark brown discoloration. Spots expanded in 2 days, and tissues collapsed after 5 days. Severely affected fruit showed cracking or nonodorous decay. Five diseased samples were collected, and causal agents were isolated from symptomatic tissues 1 cm under the peel after surface sterilization in 0.3% NaOCl for 10 min and rinsing in sterile water three times. Tissues were placed on a Luria Bertani (LB) plate for culture. Ten representative isolates were selected for further characterization. No colony was isolated from healthy tissues. Colonies were round, smooth, with irregular edges, and produced a yellow pigment in culture. Biolog identification (Version 4.20.05) showed that all strains were gram negative, negative for indole production, and utilized glucose, maltose, trehalose, sucrose, D-lactose, and pectin but not sorbitol or gelatin. The isolates were identified as Pantoea agglomerans (SIM 0.69). Multilocus sequence analysis (MLSA) was conducted for rapid classification of the strains. Sequences of atpD, gyrB, infB, and rpoB were amplified using corresponding primers (2). All sequences of the 10 isolates were identical in each gene. BLASTn was performed, and maximum likelihood trees based on the concatenated nucleotide sequences of the four genes were constructed using MEGA6. Bootstrap values after 1,000 replicates were expressed as percentages. Results showed that the tested strain named CL1 was most homologous to P. anthophila, with 98% identity for atpD (KM521543), 100% for gyrB (KM521544), infB (KM521545), and rpoB (KM521546). The 16S rRNA sequence (KM521542) amplified by primers 27f and 1492r shared 99% identity with that of P. anthophila M19_2C (JN644500). P. anthophila was previously reclassified from P. agglomerans (3); therefore, we suggest naming this wampee pathogen P. anthophila. Subsequently, 10 wampee fruits were injected with 20 μl of bacterial suspension (1 × 108 CFU/ml) of strains CL1 and CL2, respectively, and another 10 were injected with 20 μl of LB medium as controls, all kept at 28°C for 4 days. Symptoms similar to those of natural infections were observed on inoculated fruits but not on the negative controls. Bacteria were isolated from diseased tissues and further identified as P. anthophila by gyrB sequencing. P. anthophila was reported to naturally infect balsam and marigold (1,2). To our knowledge, this is the first report of P. anthophila naturally causing soft rot disease and cracking on C. lansium (wampee). References: (1) C. Brady et al. Syst. Appl. Microbiol. 31:447, 2008. (2) C. Brady et al. Int. J. Syst. Evol. Microbiol. 59:2339, 2009. (3) J. Morton. Fruits of Warm Climates. Echo Point Books & Media, Miami, FL, 1987.

Matched MeSH terms: Clausena
Fulltext Antiproliferative Properties of Clausine-B against Cancer Cell Lines

Wan Mohd Zain WN, Rahmat A, Othman F, Yap TY

Malays J Med Sci, 2009 Jul;16(3):29-34.
PMID: 22589662 MyJurnal

CLAUSINE B, A CARBAZOLE ALKALOID ISOLATED FROM THE STEM BARK OF CLAUSENA EXCAVATA, WAS INVESTIGATED FOR ITS ANTIPROLIFERATIVE ACTIVITIES AGAINST FIVE HUMAN CANCER CELL LINES: HepG2 (hepatic cancer), MCF-7 (hormone-dependent breast cancer), MDA-MB-231 (non-hormone-dependent breast cancer), HeLa (cervical cancer), and CAOV3 (ovarian cancer).

Matched MeSH terms: Clausena
Clausenidin from Clausena excavata induces apoptosis in hepG2 cells via the mitochondrial pathway

Waziri PM, Abdullah R, Yeap SK, Omar AR, Abdul AB, Kassim NK, et al.

J Ethnopharmacol, 2016 Dec 24;194:549-558.
PMID: 27729282 DOI: 10.1016/j.jep.2016.10.030

ETHNOPHARMACOLOGICAL RELEVANCE: Clausena excavata Burm.f. is used locally in folk medicine for the treatment of cancer in South East Asia.
AIM OF THE STUDY: To determine the mechanism of action of pure clausenidin crystals in the induction of hepatocellular carcinoma (hepG2) cells apoptosis.
MATERIALS AND METHODS: Pure clausenidin was isolated from Clausena excavata Burm.f. and characterized using (1)H and (13)C NMR spectra. Clausenidin-induced cytotoxicity was determined by MTT assay. The morphology of hepG2 after treatment with clausenidin was determined by fluorescence and Scanning Electron Microscopy. The effect of clausenidin on the apoptotic genes and proteins were determined by real-time qPCR and protein array profiling, respectively. The involvement of the mitochondria in clausenidin-induced apoptosis was investigated using MMP, caspase 3 and 9 assays.
RESULTS: Clausenidin induced significant (p<0.05) and dose-dependent apoptosis of hepG2 cells. Cell cycle assay showed that clausenidin induced a G2/M phase arrest, caused mitochondrial membrane depolarization and significantly (p<0.05) increased expression of caspases 3 and 9, which suggest the involvement of the mitochondria in the apoptotic signals. In addition, clausenidin caused decreased expression of the anti-apoptotic protein, Bcl 2 and increased expression of the pro-apoptotic protein, Bax. This finding was confirmed by the downregulation of Bcl-2 gene and upregulation of the Bax gene in the treated hepG2 cells.
CONCLUSION: Clausenidin extracted from Clausena excavata Burm.f. is an anti-hepG2 cell compound as shown by its ability to induce apoptosis through the mitochondrial pathway of apoptosis. Clausenidin can potentially be developed into an anticancer compound.

Matched MeSH terms: Clausena/chemistry*
Fulltext Effect of Clausena excavata Burm. f. (Rutaceae) leaf extract on wound healing and antioxidant activity in rats

Albaayit SF, Abba Y, Rasedee A, Abdullah N

Drug Des Devel Ther, 2015;9:3507-18.
PMID: 26203223 DOI: 10.2147/DDDT.S84770

Clausena excavata is a well-known plant used in folkloric medicine for the treatment of different ailments. This study aimed to determine the in vitro cytoxicity of its leaf solvent extracts as well as the in vivo wound healing and antioxidant activities of the methanolic extracts of C. excavata (MECE). HaCaT (keratocyte) and Vero cell lines were used for evaluation of the in vitro cytotoxic effects, while the in vivo wound healing and antioxidant activities were determined in skin wounds inflicted on rats. Twenty adult male Sprague-Dawley rats were divided into five groups of four animals each. Approximately 3.14 cm(2) excisional wound was inflicted on the nape of each rat following anesthesia. The treatment groups received topical application of MECE at 50 mg/mL (MECE-LD [low dose]), 100 mg/mL (MECE-MD [medium dose]), and 200 mg/mL (MECE-HD [high dose]), while the negative control group was treated with gum acacia in normal saline and the positive control group with intrasite gel. Wound contraction was evaluated on days 5, 10, and 15 after wound infliction, and tissue from wound area was collected at day 15 post-wound infliction for antioxidant enzyme evaluation and histopathological analyses. Generally, Vero cells were more resistant to the cytotoxic effects of the solvent extracts as compared with HaCaT cells. Chloroform (CH) and ethyl acetate (EA) extracts of C. excavata were toxic to HaCaT cells at 200 and 400 µg/mL, but the same concentrations showed higher (P<0.05) viability in Vero cells. There was significantly (P<0.01) greater wound contraction at days 10 and 15 post-wound infliction in all the treatment groups than in the control groups. Histopathologically, the MECE-HD-treated wound showed significantly (P<0.05) lesser inflammatory cell proliferation, degeneration, and distribution of granulation tissue than other groups. Similarly, the degree of collagen maturation, angiogenesis, and collagen distribution were significantly (P<0.05) lower in MECE-HD than in other groups. The MECE-HD, MECE-MD, and intrasite treatment groups showed a significantly (P<0.05) higher number of VEGF-positive and TGF-β1-positive cells in the skin wound than the control groups. The activities of superoxide dismutase and catalase were significantly (P<0.01) higher in the MECE-HD and intrasite treatment groups than in the other groups. Lipid peroxidase activity of the treated groups was significantly (P<0.01) lower than that in the control group. The study showed that MECE is a potent wound healing agent through anti-inflammatory and antioxidant effects that enhanced the rate of wound contraction, re-epithelialization, and collagen deposition. The effect of MECE is suggested to be due to its high polyphenolic compound content.

Matched MeSH terms: Clausena/chemistry*
Fulltext Clausenidin induces caspase-dependent apoptosis in colon cancer

Waziri PM, Abdullah R, Yeap SK, Omar AR, Kassim NK, Malami I, et al.

BMC Complement Altern Med, 2016 Jul 29;16:256.
PMID: 27473055 DOI: 10.1186/s12906-016-1247-1

BACKGROUND: Clausena excavata Burm.f. is a shrub traditionally used to treat cancer patients in Asia. The main bioactive chemical components of the plant are alkaloids and coumarins. In this study, we isolated clausenidin from the roots of C. excavata to determine its apoptotic effect on the colon cancer (HT-29) cell line.
METHOD: We examined the effect of clausenidin on cell viability, ROS generation, DNA fragmentation, mitochondrial membrane potential in HT-29 cells. Ultrastructural analysis was conducted for morphological evidence of apoptosis in the treated HT-29 cells. In addition, we also evaluated the effect of clausenidin treatment on the expression of caspase 3 and 9 genes and proteins in HT-29 cells.
RESULT: Clausenidin induced a G0/G1 cell cycle arrest in HT-29 cells with significant (p

Matched MeSH terms: Clausena/chemistry*
Clausenidin Induces Caspase 8-Dependent Apoptosis and Suppresses Production of VEGF in Liver Cancer Cells

Waziri PM, Abdullah R, Rosli R, Omar AR, Abdul AB, Kassim NK, et al.

Asian Pac J Cancer Prev, 2018 Apr 25;19(4):917-922.
PMID: 29693341

Clausena excavata Burm f. is used by traditional healers to treat cancer patients in South East Asia. The use of the
plant and its compounds is based on Asian folklore with little or no scientific evidence supporting the therapeutic efficacy
The current study aimed to determine the effect of pure clausenidin isolated from C. excavata on caspase-8-induced cell
death as well as angiogenesis in the HepG2 hepatocellular carcinoma cell line. Caspase-8 and extrinsic death receptor
protein expression was determined using spectrophotometry and protein profile arrays, respectively. Ultrastructural
analysis of clausenidin-treated cells was conducted using transmission electron microscopy. In addition, anti-angiogenic
effects of clausenidin were investigated by Western blot analysis. Clausenidin significantly (p<0.05) increased the
activity of caspase-8 and expression of protein components of the death inducing signaling complex (DISC) in HepG2
cells. Ultrastructural analysis of the clausenidin-treated HepG2 cells revealed morphological abnormalities typical of
apoptosis. Furthermore, clausenidin significantly (p<0.05) decreased the expression of vascular endothelial growth
factor (VEGF). Therefore, clausenidin is a potential anti-angiogenic agent which may induce apoptosis of hepatocellular
carcinoma cells.

Matched MeSH terms: Clausena/chemistry*
Fulltext Dentatin Induces Apoptosis in Prostate Cancer Cells via Bcl-2, Bcl-xL, Survivin Downregulation, Caspase-9, -3/7 Activation, and NF-κB Inhibition

Arbab IA, Looi CY, Abdul AB, Cheah FK, Wong WF, Sukari MA, et al.

Evid Based Complement Alternat Med, 2012;2012:856029.
PMID: 23091559 DOI: 10.1155/2012/856029

This study was set to investigate antiproliferative potential of dentatin (a natural coumarin isolated from Clausena excavata Burm. F) against prostate cancer and to delineate the underlying mechanism of action. Treatment with dentatin dose-dependently inhibited cell growth of PC-3 and LNCaP prostate cancer cell lines, whereas it showed less cytotoxic effects on normal prostate epithelial cell line (RWPE-1). The inhibitory effect of dentatin on prostate cancer cell growth was due to induction of apoptosis as evidenced by Annexin V staining and cell shrinkage. We found that dentatin-mediated accumulation of reactive oxygen species (ROS) and downregulated expression levels of antiapoptotic molecules (Bcl-2, Bcl-xL, and Survivin), leading to disruption of mitochondrial membrane potential (MMP), cell membrane permeability, and release of cytochrome c from the mitochondria into the cytosol. These effects were associated with induction of caspase-9, -3/7 activities, and subsequent DNA fragmentation. In addition, we found that dentatin inhibited TNF-α-induced nuclear translocation of p65, suggesting dentatin as a potential NF-κB inhibitor. Thus, we suggest that dentatin may have therapeutic value in prostate cancer treatment worthy of further development.

Matched MeSH terms: Clausena
Dentatin from Clausena excavata Induces Apoptosis in HepG2 Cells via Mitochondrial Mediated Signaling

Andas AR, Abdul AB, Rahman HS, Sukari MA, Abdelwahab SI, Samad NA, et al.

Asian Pac J Cancer Prev, 2015;16(10):4311-6.
PMID: 26028091

Hepatocellular carcinoma (HCC) is a primary liver cancer with high global incidence and mortality rates. Current candidate drugs to treat HCC remain lacking and those in use possess undesirable side effects. In this investigation, the antiproliferative effects of dentatin (DTN), a natural coumarin, were evaluated on HepG2 cells and DTN's probable preliminary molecular mechanisms in apoptosis induction were further investigated. DTN significantly (p<0.05) suppressed proliferation of HepG2 cells with an IC50 value of 12.0 μg/mL, without affecting human normal liver cells, WRL-68 (IC50>50 μg/mL) causing G0/G1 cell cycle arrest via apoptosis induction. Caspase colorimetric assays showed markedly increased levels of caspase-3 and caspase-9 activities throughout the treatment period. Western blotting of treated HepG2 cells revealed inhibition of NF-κB that triggers the mitochondrial-mediated apoptotic signaling pathway by up-regulating cytoplasmic cytochrome c and Bax, and down-regulating Bcl-2 and Bcl-xL. The current findings suggest DTN has the potential to be developed further as an anticancer compound targeting human HCC.

Matched MeSH terms: Clausena*
Fulltext Anti-ulcerogenic activity of dentatin from clausena excavata Burm.f. against ethanol-induced gastric ulcer in rats: Possible role of mucus and anti-oxidant effect

Sidahmed HMA, Vadivelu J, Loke MF, Arbab IA, Abdul B, Sukari MA, et al.

Phytomedicine, 2019 Mar 01;55:31-39.
PMID: 30668441 DOI: 10.1016/j.phymed.2018.06.036

BACKGROUND: Clausena excavata Burm.f. (Rutaceae) has been used for the treatment of stomach disorders including peptic ulcer.
PURPOSE: In this study, we aimed to investigate dentatin isolated from C. excavata Burm.f., for anti-ulcer activity against ethanol ulcer model in rats.
METHODS: Gastric acid output, ulcer index, serum profile, histological evaluation using Hematoxylin and eosin (HE), periodic acid Schiff base stainings and immunohistochemical localization for heat shock proteins 70 (HSP70) were all investigated. Possible involvement of reduced glutathione (GSH), lipid peroxidation, prostaglandin E2 (PGE2), superoxide dismutase (SOD) enzymes, radical scavenging, and anti-Helicobacter pylori activity were investigated.
RESULTS: Dentatin showed anti-secretory activity against the pylorus ligature model and protected the gastric mucosa from ethanol ulceration, as revealed by the improved macroscopic and histological appearance. Dentatin significantly increased the gastric homogenate content of PGE2 GSH and SOD. Dentatin inhibited the lipid peroxidation as revealed by the reduced gastric content of malondialdehyde (MDA). Moreover, dentatin up-regulated HSP70 expression. However, dentatin showed insignificant anti-H. pylori activity.
CONCLUSION: Dentatin possesses gastro-protective activity, which could be attributed to the anti-secretory, mucus production, anti-oxidant, and HSP70 activities.

Matched MeSH terms: Clausena/chemistry
Inducing G2/M Cell Cycle Arrest and Apoptosis through Generation Reactive Oxygen Species (ROS)-Mediated Mitochondria Pathway in HT-29 Cells by Dentatin (DEN) and Dentatin Incorporated in Hydroxypropyl-β-Cyclodextrin (DEN-HPβCD)

Ashwaq AS, Al-Qubaisi MS, Rasedee A, Abdul AB, Taufiq-Yap YH, Yeap SK

Int J Mol Sci, 2016 Oct 18;17(10).
PMID: 27763535

Dentatin (DEN), purified from the roots of Clausena excavata Burm f., has poor aqueous solubility that reduces its therapeutic application. The aim of this study was to assess the effects of DEN-HPβCD (hydroxypropyl-β-cyclodextrin) complex as an anticancer agent in HT29 cancer cell line and compare with a crystal DEN in dimethyl sulfoxide (DMSO). The exposure of the cancer cells to DEN or DEN-HPβCD complex leads to cell growth inhibition as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. To analyze the mechanism, in which DEN or DEN-HPβCD complex causes the death in human colon HT29 cancer cells, was evaluated by the enzyme-linked immunosorbent assay (ELIZA)-based assays for caspase-3, 8, 9, and reactive oxygen species (ROS). The findings showed that an anti-proliferative effect of DEN or DEN-HPβCD complex were via cell cycle arrest at the G2/M phase and eventually induced apoptosis through both mitochondrial and extrinsic pathways. The down-regulation of poly(ADP-ribose) polymerase (PARP) which leaded to apoptosis upon treatment, was investigated by Western-blotting. Hence, complexation between DEN and HPβCD did not diminish or eliminate the effective properties of DEN as anticancer agent. Therefore, it would be possible to resolve the conventional and current issues associated with the development and commercialization of antineoplastic agents in the future.

Matched MeSH terms: Clausena/chemistry
Dentatin isolated from Clausena excavata induces apoptosis in MCF-7 cells through the intrinsic pathway with involvement of NF-κB signalling and G0/G1 cell cycle arrest: a bioassay-guided approach

Arbab IA, Abdul AB, Sukari MA, Abdullah R, Syam S, Kamalidehghan B, et al.

J Ethnopharmacol, 2013 Jan 9;145(1):343-54.
PMID: 23178663 DOI: 10.1016/j.jep.2012.11.020

Clausena excavata Burm. f. has been used in folk medicines in eastern Thailand for the treatment of cancer.

Matched MeSH terms: Clausena/chemistry

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