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1.  Assessment of phytochemicals, antimicrobial and cytotoxic activities of extract and fractions from Fagonia olivieri (Zygophyllaceae) 
Background
In Pakistan Fagonia olivieri (Zygophyllaceae) is commonly used in the indigenous system of medicine for treatment of conditions like diabetes, cancer, fever, asthma, toothache, stomach troubles and kidney disorders. This study evaluated the crude methanol extract of F. olivieri (FOM) and its derived fractions for their antimicrobial and cytotoxic activities as well as the classes of phytochemical.
Methods
Dried powder of whole plant of F. olivieri was extracted with methanol (FOM) and the resultant was fractionated to give n-hexane fraction (FOH), chloroform fraction (FOC), ethyl acetate fraction (FOE), n-butanol fraction (FOB) and residual aqueous fraction (FOA). Methanol extract and its derived fractions were subjected to phytochemical screening using standard procedures. Also the extract and fractions were assayed for antibacterial, antifungal and cytotoxic activities using agar well diffusion technique, agar tube dilution method and brine shrimps lethality test, respectively.
Results
The results obtained for phytochemical analysis indicate the presence of saponins and alkaloids in all the tested extract and fractions while anthraquinones were not detected. The results showed that all the bacterial strains tested in this study were susceptible to at least one of the fractions tested. However, FOE and FOB were the best antibacterial fractions and showed antibacterial activity against maximum number of bacterial strains. The results showed that Escherichia coli was the most sensitive bacterium while Bordetella bronchiseptica and Enterobacter aerogenes were less susceptible against various fractions. Maximum percent inhibition for growth was recorded for the fungus Aspergillus flavus with FOE whereas growth of Aspergillus fumigatus and Fusarium solani was inhibited by FOM and its all derived fractions. Minimum LC50 (24.07 mg/L) for brine shrimp assay was recorded for FOE followed by LC50 of FOC (26.1 mg/L) and FOB (30.05 mg/L) whereas maximum LC50 was exhibited by FOH (1533 mg/L).
Conclusion
These results indicated the use of F. olivieri to treat infections with emphasis to isolate and characterize the active principle responsible for antibacterial, antifungal and cytotoxic activities and its exploitation as therapeutic agent.
doi:10.1186/1472-6882-13-167
PMCID: PMC3717109  PMID: 23842440
Antibacterial activity; Brine shrimps lethality assay; Cytotoxicity; Fagonia olivieri; Phytochemicals
2.  Evaluation of pharmacological activities, cytotoxicity and phenolic composition of four Maytenus species used in southern African traditional medicine to treat intestinal infections and diarrhoeal diseases 
Background
Microbial infections and resulting inflammation and oxidative stress are common pathogenesis of gastrointestinal tract (GIT) disorders. In South Africa, several species of the genus Maytenus are used in traditional medicine to treat various infectious diseases. Most of the previous work on this genus was focused on nonpolar extracts from the root and bark. In this study, leaf extracts of polar extracts of Maytenus peduncularis, Maytenus procumbens, Maytenus senegalensis and Maytenus undata were evaluated for antimicrobial, anti-inflammatory and antioxidant activities to determine their efficacy as therapeutic agents in GIT disorders.
Methods
Phenolic-enriched leaf extracts and fractions were prepared by extracting with acidified 70% methanol and solvent-solvent fractionation. The activities of the fractions against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus faecalis as well as clinical isolates of Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans were determined using a serial microplate dilution method. Antioxidant activities were determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), hydroxyl (OH) radical scavenging and linoleic acid peroxidation inhibitory assays. The phenolic composition as well as the cytotoxicity against Vero cell lines of the crude extracts was evaluated using various standard protocols.
Results
The antimicrobial activities were concentrated in the non-polar fractions of hexane, dichloromethane and ethyl acetate (MICs 19–312 μg/ml). The crude extracts and polar fractions (butanol and water) had moderate to poor antimicrobial activity (MICs 312 to above 2500 μg/ml). The crude extracts and polar fractions had good antioxidant activity (EC50 values varied from 1.22 to 607 μg/ml, 1.71 to 312 μg/ml and 23 to 284 μg/ml for DPPH, ABTS and OH respectively. Linoleic acid peroxidation inhibition EC50 values of the crude extracts ranged between 27 and 39 μg/ml with relatively low toxicity against Vero cell lines (IC50 values 87 to 187 μg/ml). Fractionation of a crude extract with low activity could lead to fractions with more potent activity.
Conclusion
This study justifies the traditional use of leaf crude extracts and fractions from these four plants to remedy gastrointestinal disorders resulting from infection, inflammation and oxidative stress complications. The study also provides rationale for the use of leaf extracts with same beneficial effects in place of unsustainable root and bark harvest.
doi:10.1186/1472-6882-13-100
PMCID: PMC3726504  PMID: 23663902
Maytenus; Gastrointestinal tract; Infections; Diarrhoea; Cytotoxicity
3.  Antibacterial, antidiarrhoeal, and cytotoxic activities of methanol extract and its fractions of Caesalpinia bonducella (L.) Roxb leaves 
Background
Caesalpinia bonducella is an important medicinal plant for its traditional uses against different types of diseases. Therefore, the present study investigated the antimicrobial, antidiarrhoeal, and cytotoxic activities of the methanol extract and ethyl acetate, chloroform, and petroleum ether (pet. ether) fractions of C. bonducella leaves.
Methods
The antibacterial potentialities of methanol extract and its fractions of C. bonducella leaves were investigated by the disc diffusion method against four gram-positive and five gram-negative bacteria at 300, 500 and 800 μg/disc. Kanamycin (30 μg/disc) was used as the standard drug. Antidiarrhoeal activities of leaf extracts were evaluated at two doses (200 and 400 mg/kg) and compared with loperamide in a castor oil-induced diarrhoeal model in rat. The fractions were subjected to a brine shrimp lethality test to evaluate their cytotoxicity.
Results
The methanol extract and other three fractions exhibited better activities at higher concentrations. Amongst, the chloroform fraction showed maximum activity at all three concentrations (300, 500, and 800 μg/disc) against almost all bacteria. S. aureus and P. aeruginosa showed better sensitivities to all extracts at all three concentrations excluding the pet. ether fraction. Bacillus megaterium and Klebsiella spp. were two bacteria amongst nine that showed lowest sensitivity to the extracts. Maximum zone of inhibition (25-mm) was obtained by the methanol extract at an 800 μg/disc concentration against S. aureus. In the antidiarrhoeal test, all fractions exhibited dose-dependent actions, which were statistically significant (p < 0.05). Ethyl acetate fraction exerted maximum inhibition (51.11%) against defecation, whereas 57.75% inhibition was obtained for loperamide. Moderate cytotoxicity was found for the methanol extract and its three fractions compared with the standard drug vincristine sulfate in the brine shrimp bioassay. In the present study, the LC50 values of the methanol crude extract and ethyl acetate, chloroform, pet. ether fractions and vincristine sulfate were 223.87, 281.84, 112.2, 199.53, and 12.59 μg/mL, respectively. Therefore, the ethyl acetate fraction showed maximum cytotoxicity, whereas minimum cytotoxicity was observed for the chloroform fraction.
Conclusion
The present study revealed that the ethyl acetate fraction of the C. bonducella leaves has significant antidiarrhoeal properties. The methanol extract and other three fractions of the C. bonducella leaves possess potent antibacterial activities along with moderate cytotoxicities that may lead to new drug development.
doi:10.1186/1472-6882-13-101
PMCID: PMC3661353  PMID: 23663985
Caesalpinia bonducella; Antimicrobial; Antidiarrhoeal; Cytotoxicity
4.  Antimicrobial Activity and Brine Shrimp Lethality Bioassay of the Leaves Extract of Dillenia indica Linn 
The crude methanolic extract of Dillenia indica Linn. (Dilleniaceae) leaves has been investigated for the evaluation of antimicrobial and cytotoxic activities. Organic solvent (n-hexane, carbon tetrachloride and chloroform) fractions of methanolic extract and methanolic fraction (aqueous) were screened for their antimicrobial activity by disc diffusion method. Besides, the fractions were screened for cytotoxic activity using brine shrimp (Artemia salina) lethality bioassay. Among the four fractions tested, n-hexane, carbon tetrachloride, and chloroform fractions showed moderate antibacterial and antifungal activity compared to standard antibiotic, kanamycin. The average zone of inhibition was ranged from 6 to 8 mm at a concentration of 400 µg/disc. But the aqueous fraction was found to be insensitive to microbial growth. Compared to vincristine sulfate (with LC50 of 0.52 µg/ ml), n-hexane and chloroform fractions demonstrated a significant cytotoxic activity (having LC50 of 1.94 µg/ml and 2.13 µg/ml, respectively). The LC50 values of the carbon tetrachloride and aqueous fraction were 4.46 µg/ml and 5.13 µg/ ml, respectively. The study confirms the moderate antimicrobial and potent cytotoxic activities of Dillenia indica leaves extract and therefore demands the isolation of active principles and thorough bioassay.
doi:10.4103/0975-1483.62213
PMCID: PMC3035885  PMID: 21331191
Antimicrobial activity; Artemia salina; brine shrimp lethality bioassay; Dillenia indica
5.  Allelopathic Effects of Water Hyacinth [Eichhornia crassipes] 
PLoS ONE  2010;5(10):e13200.
Eichhornia crassipes (Mart) Solms is an invasive weed known to out-compete native plants and negatively affect microbes including phytoplankton. The spread and population density of E. crassipes will be favored by global warming. The aim here was to identify compounds that underlie the effects on microbes. The entire plant of E. crassipes was collected from El Zomor canal, River Nile (Egypt), washed clean, then air dried. Plant tissue was extracted three times with methanol and fractionated by thin layer chromatography (TLC). The crude methanolic extract and five fractions from TLC (A–E) were tested for antimicrobial (bacteria and fungal) and anti-algal activities (green microalgae and cyanobacteria) using paper disc diffusion bioassay. The crude extract as well as all five TLC fractions exhibited antibacterial activities against both the Gram positive bacteria; Bacillus subtilis and Streptococcus faecalis; and the Gram negative bacteria; Escherichia coli and Staphylococcus aureus. Growth of Aspergillus flavus and Aspergillus niger were not inhibited by either E. crassipes crude extract nor its five fractions. In contrast, Candida albicans (yeast) was inhibited by all. Some antialgal activity of the crude extract and its fractions was manifest against the green microalgae; Chlorella vulgaris and Dictyochloropsis splendida as well as the cyanobacteria; Spirulina platensis and Nostoc piscinale. High antialgal activity was only recorded against Chlorella vulgaris. Identifications of the active antimicrobial and antialgal compounds of the crude extract as well as the five TLC fractions were carried out using gas chromatography combined with mass spectroscopy. The analyses showed the presence of an alkaloid (fraction A) and four phthalate derivatives (Fractions B–E) that exhibited the antimicrobial and antialgal activities.
doi:10.1371/journal.pone.0013200
PMCID: PMC2951916  PMID: 20949048
6.  Interleukin-6 and Cyclooxygenase-2 downregulation by fatty-acid fractions of Ranunculus constantinopolitanus 
Background
Medicinal plants represent alternative means for the treatment of several chronic diseases, including inflammation. The genus Ranunculus, a representative of the Ranunculaceae family, has been reported to possess anti-inflammatory, analgesic, antiviral, antibacterial, antiparasitic and antifungal activities, possibly due to the presence of anemonin and other. Different studies have shown the occurrence of unusual fatty acids (FAs) in Ranunculaceae; however, their therapeutic role has not been investigated. The purpose of this study is to characterize potential anti-inflammatory bioactivities in Ranunculus constantinopolitanus D'Urv., traditionally used in Eastern Mediterranean folk medicine.
Methods
The aerial part of R. constantinopolitanus was subjected to methanol (MeOH) extraction and solvent fractionation. The bioactive fraction (I.2) was further fractionated using column chromatography, and the biologically active subfraction (Y2+3) was identified using infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). The effects of I.2 and Y2+3 on cell viability were studied in mouse mammary epithelial SCp2 cells using trypan blue exclusion method. To study the anti-inflammatory activities of I.2 and Y2+3, their ability to reduce interleukin (IL)-6 levels was assessed in endotoxin (ET)-stimulated SCp2 cells using enzyme-linked immunosorbent assay (ELISA). In addition, the ability of Y2+3 to reduce cyclooxygenase (COX)-2 expression was studied in IL-1-treated mouse intestinal epithelial Mode-K cells via western blotting. Data were analyzed by one-way analysis of variance (ANOVA), Student-Newman-Keuls (SNK), Tukey HSD, two-sample t-test and Dunnett t-tests for multiple comparisons.
Results
The chloroform fraction (I.2) derived from crude MeOH extract of the plant, in addition to Y2+3, a FA mix isolated from this fraction and containing palmitic acid, C18:2 and C18:1 isomers and stearic acid (1:5:8:1 ratio), reduced ET-induced IL-6 levels in SCp2 cells without affecting cell viability or morphology. When compared to fish oil, conjugated linoleic acid (CLA) and to individual FAs as palmitic, linoleic, oleic and stearic acid or to a mix of these FAs (1:5:8:1 ratio), Y2+3 exhibited higher potency in reducing ET-induced IL-6 levels within a shorter period of time. Y2+3 also reduced COX-2 expression in IL-1-treated Mode-K cells.
Conclusion
Our studies demonstrate the existence of potential anti-inflammatory bioactivities in R. constantinopolitanus and attribute them to a FA mix in this plant.
doi:10.1186/1472-6882-9-44
PMCID: PMC2784432  PMID: 19917107
7.  Antioxidant and Antimicrobial activity of Pedicularis sibthorpii Boiss. And Pedicularis wilhelmsiana Fisch ex.  
Purpose: This research paper presents antioxidant and antimicrobial activities of Pedicularis sibthorpii and Pedicularis wilhelmsiana which grow in Azerbaijan/Iran with claimed a lot of therapeutic effects. Methods: DPPH assay and agar well diffusion method were carried out to determine antioxidant and antimicrobial activities respectively. Results: Methanolic extract showed better antioxidant activity compared to other crude extracts (n-hexane and dichloromethane). Methanolic extracts of both Pedicularis sibthorpii and Pedicularis wilhelmsiana were found to have antibacterial activity especially against gram positive strains of S. ureus, S.epidermidis. No antifungal activity was observed in the tested extracts. Conclusion: Existence of some phenolic compounds in methanolic extracts, such as phenylethanoids and flavonoids (found in other species of Pedicularis), which cause both antioxidant and antibacterial activities, is probable. Antimicrobial and antioxidant activity of the methanolic extracts supports further studies related to phytochemical investigation and bioassay of different fractions to isolate pure compounds of plants.
doi:10.5681/apb.2012.012
PMCID: PMC3846006  PMID: 24312775
Pedicularis sibthorpii; Pedicularis wilhelmsiana; Antimicrobial; Antioxidant; Methanolic extract
8.  In vitro antimicrobial activity of ethanolic fractions of Cryptolepis sanguinolenta 
Background
Following claims that some plants have antimicrobial activities against infectious microbes, the in vitro antimicrobial activities of different solvent fractions of ethanolic extract of Cryptolepis sanguinolenta were evaluated against eight standard bacteria and clinical isolates.
Methods
The solvent partitioning protocol involving ethanol, petroleum ether, chloroform, ethyl acetate and water, was used to extract various fractions of dried pulverized Cryptolepis sanguinolenta roots. Qualitative phyto-constituents screening was performed on the ethanol extract, chloroform fraction and the water fraction. The Kirby Bauer disk diffusion method was employed to ascertain the antibiogram of the test organisms while the agar diffusion method was used to investigate the antimicrobial properties of the crude plant extracts. The microplate dilution method aided in finding the MICs while the MBCs were obtained by the method of Nester and friends. The SPSS 16.0 version was used to analyze the percentages of inhibitions and bactericidal activities.
Results
The phytochemical screening revealed the presence of alkaloids, reducing sugars, polyuronides, anthocyanosides and triterpenes. The ethanol extract inhibited 5 out of 8 (62.5%) of the standard organisms and 6 out of 8 (75%) clinical isolates. The petroleum ether fraction inhibited 4 out of 8 (50%) of the standard microbes and 1 out of 8 (12.5%) clinical isolates. It was also observed that the chloroform fraction inhibited the growth of all the organisms (100%). Average inhibition zones of 14.0 ± 1.0 mm to 24.67 ± 0.58 mm was seen in the ethyl acetate fraction which halted the growth of 3 (37.5%) of the standard organisms. Inhibition of 7 (87.5%) of standard strains and 6 (75%) of clinical isolates were observed in the water fraction. The chloroform fraction exhibited bactericidal activity against all the test organisms while the remaining fractions showed varying degrees of bacteriostatic activity.
Conclusion
The study confirmed that fractions of Cryptolepis sanguinolenta have antimicrobial activity. The chloroform fraction had the highest activity, followed by water, ethanol, petroleum ether and ethyl acetate respectively. Only the chloroform fraction exhibited bactericidal activity and further investigations are needed to ascertain its safety and prospects of drug development.
doi:10.1186/1476-0711-11-16
PMCID: PMC3473295  PMID: 22709723
9.  Screening and evaluation of antioxidant, antimicrobial, cytotoxic, thrombolytic and membrane stabilizing properties of the methanolic extract and solvent-solvent partitioning effect of Vitex negundo Bark 
Objective
To investigate different biological activities of the methanolic extract and solvent-solvent partitioning of Vitex negundo (V. negundo) bark.
Methods
In-vitro anti-oxidant study was determined using total phenolic compound analysis and DPPH radical scavenging assay. In vitro antimicrobial study was measured by observing zone of inhibition. The cytotoxic activity was studied using brine shrimp lethality bioassay and thrombolytic activity by clot disruption method.
Results
In the evaluation of anti-oxidant activity, the amount of total phenolic content differed in different extractives as from 12.94 mg of GAE/g of extractives to 54.19 mg of GAE/g of extractives of V. negundo. Among all extractives, the highest phenolic content was found in n-hexane fraction (N-HXN) (54.19 mg of GAE/g of extractives). Significant amount of phenolic compounds were present in methanol extract (ME) (35.19 mg of GAE/g of extractives) and aqueous soluble fraction (AQSF) (12.94 mg of GAE/g of extractives). The free radical scavenging activity of ME and its different partitions were assessed using DPPH free radicals assay. The highest free radical scavenging activity was found in AQSF (IC50=7.78 µg/mL). The antimicrobial screening of the bark of V. negundo exhibited mild to moderate activity in test microorganisms. The chloroform soluble fraction demonstrated the highest inhibition against microbial growth having zone of inhibition as 8-12 mm. In the brine shrimp lethality bioassay, The LC50 values of n-hexane and chloroform soluble fractions were found 3.713 6 µg/mL, and 0.910 µg/mL respectively while the LC50 values of standard Vincristine sulphate was 0.408 µg/mL. The methanol extract and their different organic soluble of V. negundo bark inhibited hypotonic solution-induced haemolysis of erythrocytes in in-vitro membrane stabilizing activity test. The AQSF and N-HXN showed inhibition of haemolysis as 55.05% and 29.89% that were lower than 71.9% of aspirin (0.10 mg/mL). Methanol extract of V. negundo and all of its different partitions exhibited moderate thrombolytic activity of 36.95%-22.98%.
Conclusions
These experiments were able to show the several biological activity of methanolic extract and its soluble fractions of V. negundo at a time.
doi:10.1016/S2222-1808(13)60090-0
PMCID: PMC4027319
Vitex negundo; Antioxidant; Antimicrobial; Cytotoxic; Thrombolytic
10.  Antimicrobial and antioxidant properties of methanol extract, fractions and compounds from the stem bark of Entada abyssinica Stend ex A. Satabie 
Background
The aim of this study was to evaluate the antimicrobial and antioxidant activities of the methanol extract, fractions and isolated compounds from Entada abyssinica stem bark, plant used traditionally against gastrointestinal infections.
Methods
The methanol extract of E. abyssinica stem bark was pre-dissolved in a mixture of methanol and water, and then partitioned between n-hexane, ethyl acetate and n-butanol. The ethyl acetate portion was fractionated by column chromatography and the structures of isolated compounds elucidated by analysis of spectroscopic data and comparison with literature data. Antimicrobial activity was assayed by broth microdilution techniques on bacteria and yeasts. The antioxidant activity was determined by DPPH radical scavenging method.
Results
Four known compounds [(5S,6R,8aR)-5-(carboxymethyl)-3,4,4a,5,6,7,8,8a-octahydro-5,6,8a-trimethylnaphthalenecarboxylic acid (1), methyl 3,4,5-trihydroxybenzoate (2), benzene-1,2,3-triol (3) and 2,3-dihydroxypropyltriacontanoate (4)] were isolated. Compared to the methanol extract, fractionation increased the antibacterial activities of the n-hexane and ethyl acetate fractions, while the antifungal activities increased in ethyl acetate, n-butanol and aqueous residue fractions. The isolated compounds were generally more active on bacteria (9.7 to 156.2 μg/ml) than yeasts (78.1 to 312.5 μg/ml). Apart from compound 1, the three others displayed DPPH· scavenging activity (RSa), with RSa50 values of 1.45 and 1.60 μg/ml.
Conclusion
The results obtained from this study support the ethnomedicinal use of E. abyssinica in the treatment of gastrointestinal infections and the isolated compounds could be useful in the standardisation of antimicrobial phytomedicine from this plant.
doi:10.1186/1472-6882-11-57
PMCID: PMC3157447  PMID: 21771305
11.  A study of antimicrobial activity, acute toxicity and cytoprotective effect of a polyherbal extract in a rat ethanol-HCl gastric ulcer model 
BMC Research Notes  2012;5:546.
Background
The decoction of the aerial parts of Rhynchosia recinosa (A.Rich.) Bak. [Fabaceae] is used in combination with the stem barks of Ozoroa insignis Del. (Anacardiaceae), Maytenus senegalensis (Lam.) Excell. [Celastraceae] Entada abyssinica Steud. ex A.Rich [Fabaceae] and Lannea schimperi (Hochst.)Engl. [Anacardiaceae] as a traditional remedy for managing peptic ulcers. However, the safety and efficacy of this polyherbal preparation has not been evaluated. This study reports on the phytochemical profile and some biological activities of the individual plant extracts and a combination of extracts of the five plants.
Methods
A mixture of 80% ethanol extracts of R. recinosa, O. insignis, M. senegalensis, E. abyssinica and L. schimperi at doses of 100, 200, 400 and 800 mg/kg body wt were evaluated for ability to protect Sprague Dawley rats from gastric ulceration by an ethanol-HCl mixture. Cytoprotective effect was assessed by comparison with a negative control group given 1% tween 80 in normal saline and a positive control group given 40 mg/kg body wt pantoprazole. The individual extracts and their combinations were also tested for antibacterial activity against four Gram negative bacteria; Escherichia coli (ATCC 25922), Salmonella typhi (NCTC 8385), Vibrio cholerae (clinical isolate), and Klebsiella pneumoniae (clinical isolate) using the microdilution method. In addition the extracts were evaluated for brine shrimp toxicity and acute toxicity in mice. Phytochemical tests were done using standard methods to determine the presence of tannins, saponins, steroids, cardiac glycosides, flavonoids, alkaloids and terpenoids in the individual plant extracts and in the mixed extract of the five plants.
Results
The combined ethanolic extracts of the 5 plants caused a dose-dependent protection against ethanol/HCl induced ulceration of rat gastric mucosa, reaching 81.7% mean protection as compared to 87.5% protection by 40 mg/kg body wt pantoprazole. Both the individual plant extracts and the mixed extracts of 5 plants exhibited weak to moderate antibacterial activity against four G-ve bacteria. Despite Ozoroa insignis being toxic to mice at doses above 1000 mg/kg body wt, the other plant extracts and the combined extract of the 5 plants were tolerated by mice up to 5000 mg/kg body wt. The brine shrimp test results showed the same pattern of toxicity with Ozoroa insignis being the most toxic (LC50 = 10.63 μg/ml). Phytochemical tests showed that the combined extract of the five plants contained tannins, saponins, steroids, cardiac glycosides, flavonoids and terpenoids. Flavonoids, tannins and terpenoids are known to have antioxidant activity.
Conclusion
The combined extract of the five plants exhibited a dose-dependent protective activity in the rat ethanol-HCl gastric ulcer model. The extracts also exhibited weak antibacterial activity against four Gram negative bacteria and low acute toxicity in mice and brine shrimps. Although the results support claims by traditional healers who use a decoction of the five plants for treatment of peptic ulcers, more models of gastric ulceration and proper animal toxicity studies are needed to validate possible clinical use of the polyherbal extract. It is also evident that the doses of the crude extracts showing protection of the gastric mucosa are too large for realistic translation to direct clinical application, but further studies using bioassay guided fractionation are important to either identify more practical fractions or active compound/s.
doi:10.1186/1756-0500-5-546
PMCID: PMC3532137  PMID: 23031266
Ozoroa insignis; Maytenus senegalensis; Entada abyssinica; Lannea schimperi; Gastroprotection; Toxicity
12.  Acute toxicity, brine shrimp cytotoxicity and relaxant activity of fruits of callistemon citrinus curtis 
Background
Callistemon citrinus Curtis belongs to family Myrtaceae that has a great medicinal importance. In our previous work, fruits of Callistemon citrinus were reported to have relaxant (antispasmodic) activity. The current work describes the screening of fractions of the crude methanol extract for tracing spasmolytic constituents so that it shall help us for isolation of bioactive compounds. Acute toxicity and brine shrimp cytotoxicity of crude methanol extract are also performed to standardize it.
Methods
The crude methanol extract was obtained by maceration with distilled water (500 ml) three times and fractionated successively with n-hexane, chloroform, ethyl acetate and n-butanol (300 ml of each solvent). Phytochemical analysis for crude methanol extract was performed. Acute toxicity studies were performed in mice. Brine shrimp cytotoxicity studies were performed to determine its cytotoxicity and standardize it. In other series of experiments, rabbits' jejunum preparations were used in screening for possible relaxant activities of various fractions. They were applied in concentrations of 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 5.0 and 10.0 mg/ml on spontaneous rabbits' jejunum preparations. In similar fashion, fractions were also tested on KCl (80 mM) -induced contractions. Calcium chloride curves were constructed in K-rich Tyrode's solution. The effects of various fractions were tested on calcium chloride curves at concentrations 1.0, 3.0, 5.0 and 10.0 mg/ml. Curves of verapamil used as reference drug at concentration 0.1 μM and 0.3 μM were also constructed. The curves were compared with their respective controls for possible right shift.
Results
Methanol extract tested strongly positive for saponins and tannins. However, it tested mild positive for presence of proteins, amino acids, carbohydrates and phenolic compounds. LD50 value for crude methanol extract is 476.25 ± 10.3 (470-481, n = 4) mg/ml. Similarly, EC50 value for brine shrimp cytotoxicity is 65.5 ± 7.28 (60.8- 69.4, n = 4) mg/ml. All the fractions relaxed the spontaneous and KCl-induced contractions. EC50 values (mg/ml) for effects of ethyl acetate fraction on spontaneous and KCl induced contractions are 2.62 ± 0.78 (2.15-3.0, n = 4) and 3.72 ± 0.86 (3.38-4.28, n = 4) respectively. Respective EC50 values (mg/ml) for n-butanol fraction are 3.59 ± 0.2(3.07-3.9, n = 4) for spontaneous, and 5.57 ± 0.2 (5.07-6.11, n = 4) for KCl- induced contractions. EC50 value for control calcium chloride curve (without extract) is -2.73 ± 0.19 (-2.6 - -2.81, n = 4) while EC50 for curves treated with 5.0 mg/ml of chloroform is -2.22 ± 0.02 (-2.16 - -2.3, n = 4). EC50 value for ethyl acetate treated (1.0 mg/ml) tissues is -1.95 ± 0.10 (-1.88 - -2.0, n = 4) vs. control EC50 = -2.71 ± 0.08 (-2.66 - -2.76, n = 4). All the fractions, except n-hexane, showed a right shift like that of verapamil (EC50 = -1.72 ± 0.15 (-1.62 - -1.8, n = 4) vs. Control EC50 = -2.41 ± 0.06 (-2.38 - - 2.44, n = 4), a standard drug that blocks voltage operated calcium channels.
Conclusion
Relaxant constituents were more concentrated in ethylacetate fraction followed by chloroform, n -butanol and aqueous fractions that warrant for its isolation. The crude methanol extract is safe at concentration 250 mg/ml or below and results of brine shrimp cytotoxicity assay imply the plant specie may be a source of cytotoxic agents.
doi:10.1186/1472-6882-11-99
PMCID: PMC3216858  PMID: 22024247
13.  Acaricidal effect of an isolate from Hoslundia opposita vahl against Amblyomma variegatum (Acari: Ixodidae) 
Pharmacognosy Research  2011;3(3):185-188.
Background:
Hoslundia opposita Vahl. (Lamiaceae), a common local shrub in Ghana, is traditionally known not only for its pharmacological benefits but also for its insecticidal properties. Its acaricidal property, however, has not been investigated.
Objective:
To test the acaricidal effects of the crude extract and fractions of H. opposita leaves as well as to isolate and characterize the acaricidal principles.
Materials and Methods:
The crude methanolic extract, pet. ether, ethyl acetate and aqueous fractions of the leaves of H. opposita were tested against the larvae of the cattle tick, Amblyomma variegatum, using the Larval Packet Test. A bioassay-guided isolation was carried out to identify the acaricidal principle obtained from the ethyl acetate fraction.
Results:
The active principle was characterised as ursolic acid, a triterpene previously isolated from the leaves of the same plant. The extract and fractions were less potent than the control, malathion (LC50 1.14 × 10-4 mg/ml). Among the plant samples however the crude methanolic extract exhibited the highest effect against the larvae (LC50 5.74 × 10-2 mg/ml), followed by the ethyl acetate fraction (LC50 8.10 × 10-2 mg/ml). Ursolic acid, pet. ether and aqueous fractions however showed weak acaricidal effects with LC50 values of 1.13 mg/ml, 8.96 × 10-1 mg/ml and 1.44 mg/ml, respectively.
Conclusion:
Ursolic acid was not as potent as the crude methanolic extract and the ethyl acetate fraction from which it was isolated. The overall acaricidal effect of H. opposita may have been due to synergy with other principles having acaricidal properties.
doi:10.4103/0974-8490.85004
PMCID: PMC3193619  PMID: 22022167
Acaricide; Larval Packet Test; ticks; ursolic acid
14.  Antioxidant, Antimicrobial and Phytochemical Analysis of Cichoriumintybus Seeds Extract and Various Organic Fractions 
This study was carried out to evaluate the antimicrobial and antioxidant effectiveness of methanolic extract and different fractions (n-butanol, ethyl acetate, chloroform and n-hexane) of C.intybus seeds. The antimicrobial activity was determined by the disc diffusion method and minimum inhibitory concentration (MIC) against a panel of microorganisms (four bacterial strains, i.e. P. multocida, E. coli, B. subtilis and S. aureus and three fungal strains, i.e A. flavus, A. niger and R. solani). The results indicated that seeds extract and fractions of C. intybus showed moderate activity as antibacterial agent. While Antifungal activity of C. intybus seeds extract/fractions was very low against A. flavus and A. niger while mild against R. solani. The C.intybus seeds extract/fractions contained appreciable levels of total phenolic contents (50.8-285 GAE mg/100g of Dry plant matter) and total flavonoid contents (43.3-150 CE mg/100g of Dry plant matter). The C. intybus seed extract/fractions also exhibited good DPPH radical scavenging activity, with IC50 ranging from 21.28-72.14 μg/mL. Of the C .intybus seeds solvent extract/fractions tested, 100% methanolic extract and ethylacetate fraction exhibited the maximum antioxidant activity. The results of the present investigation demonstrated significant (p < 0.01) variations in the antioxidant and antimicrobial activities of C. intybus seeds solvent extract/fractions.
PMCID: PMC3813158  PMID: 24250548
C.intybus; Antimicrobial activity; Resazurin; A. flavus; Minimum inhibitory concentration
15.  Activity-guided isolation and identification of anti-staphylococcal components from Senecio tenuifolius Burm. F. leaf extracts 
Objective
To investigate activity-guided isolation and identification of anti-Staphylococcus aures components from Senecio tenuifolius Burm. F. (S. tenuifolius).
Methods
Hexane, chloroform, ethyl acetate, methanol and aqueous extracts of S. tenuifolius were prepared by soxilation for antimicrobial activity against one registered Staphylococcus aureus (S. aureus) (ATCC No: 25923) and two clinical isolates, methicillin resistant and methicillin sensitive S. aureus. NCCL standard methods were followed for antibacterial activity. GC-MS was performed to identify the chemical composition of bio active fraction.
Results
Among all solvent extracts, methanol extract significantly reduced the growth of S. aureus (ATCC No: 25923), methicillin resistant and methicillin sensitive S. aureus with the best zone of inhibition at 16.23, 14.06 and 15.23 mm and minimum inhibition concentration (MIC) values at 426.16, 683.22 and 512.12 µg/mL, respectively. In order to detect the active component in methanol extract, it was further purified by column chromatography, which yielded four fractions (St1, St2, St3, and St4). Among these four fractions, St3 was effective against the tested strains of S. aures, with the best zone of inhibition at 15.09, 13.25 and 14.12 mm and with best MIC values at 88.16, 128.11 and 116.12 µg/mL, respectively. Effective fraction partially purified from S. tenuifolius (St3) yielded MIC's that were at least 20 fold less when compared to crude extract. GC-MS analysis of St3 revealed the presence of 3-[methyl-6,7-dihydro benzofuran-4 (5H)-one], 1,2-benzenedicarboxylic acid, hydroquinone, methyl ester and 3 unknown compounds.
Conclusions
The study provides scientific evidence for traditional and folklore medicinal use of S. tenuifolius in skin infections treatment.
doi:10.1016/S2221-1691(13)60048-9
PMCID: PMC3631748  PMID: 23620836
Senecio tenuifolius Burm. F.; Staphylococcus aureus; Chemical composition; GC-MS; Skin infection; Topical application
16.  Antimicrobial and antioxidant activities of the extracts and compounds from the leaves of Psorospermum aurantiacum Engl. and Hypericum lanceolatum Lam. 
Background
Psorospermun aurantiacum and Hypericum lanceolatum are plants locally used in Cameroon and other parts of Africa for the treatment of gastrointestinal and urinary tract infections, skin infections, venereal diseases, gastrointestinal disorder, infertility, epilepsy as well as microbial infections. The present study was designed in order to investigate the in vitro antimicrobial and radical scavenging activities of the extracts and isolated compounds from the leaves of these plants.
Methods
The plant extract was prepared by maceration in ethyl acetate and methanol and fractionated by column chromatography. The structures of isolated compounds were elucidated by spectroscopic analyses in conjunction with literature data. The broth microdilution method was used to evaluate the in vitro antimicrobial activity against bacteria, yeasts and dermatophytes. The antioxidant potentials of the extracts and their isolated compounds were evaluated using the DPPH radical scavenging method.
Results
Five known compounds: physcion (1), 1,8-dihydroxy-3-geranyloxy-6-methylanthraquinone (2), kenganthranol B (3), vismiaquinone (4), and octacosanol (5) were isolated from the leaves of P. aurantiacum while six compounds including friedelin (6), betulinic acid (7), 2,2’,5,6’-tetrahydroxybenzophenone (8), allanxanthone A (9), 1,3,6- trihydroxyxanthone (10) and isogarcinol (11) were isolated from H. lanceolatum. Compound 8 and 4 exhibited the highest antibacterial and antifungal activities with MIC ranges of 2–8 μg/ml and 4–32 μg/ml respectively. P. aurantiacum crude extract (Rsa50 = 6.359 ± 0.101) showed greater radical scavenging activity compared with H. lanceolatum extract (Rsa50 = 30.996 ± 0.879). Compound 11 showed the highest radical scavenging activity (RSa50 = 1.012 ± 0.247) among the isolated compounds, comparable to that of L-arscobic acid (RSa50 = 0.0809 ± 0.045).
Conclusions
The experimental findings show that the ethyl acetate and methanol extracts and isolated compounds from P. aurantiacum and H. lanceolatum stem bark possess significant antimicrobial and antioxidant activities justifying the use of these plants in traditional medicine, which may be developed as phytomedicines.
doi:10.1186/1472-6882-12-136
PMCID: PMC3576290  PMID: 22916964
17.  Antimicrobial activity and brine shrimp toxicity of extracts of Terminalia brownii roots and stem 
Background
Ternimalia brownii Fresen (Combretaceae) is widely used in traditional medicine to treat bacterial, fungal and viral infections. There is a need to evaluate extracts of this plant in order to provide scientific proof for it's wide application in traditional medicine system.
Methods
Extraction of stem bark, wood and whole roots of T. brownii using solvents of increasing polarity, namely, Pet ether, dichloromethane, dichloromethane: methanol (1:1), methanol and aqua, respectively, afforded dry extracts. The extracts were tested for antifungal and antibacterial activity and for brine shrimp toxicity test.
Results
Extracts of the stem bark, wood and whole roots of T. brownii exhibited antibacterial activity against standard strains of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, and Bacillus anthracis and the fungi, Candida albicans and Cryptococcus neoformans. Aqueous extracts exhibited the strongest activity against both bacteria and fungi. Extracts of the roots and stem bark exhibited relatively mild cytotoxic activity against brine shrimp larvae with LC50 values ranging from 113.75–4356.76 and 36.12–1458.81 μg/ml, respectively. The stem wood extracts exhibited the highest toxicity against the shrimps (LC50 values 2.58–14.88 μg/ml), while that of cyclophosphamide, a standard anticancer drug, was 16.33 (10.60–25.15) μg/ml.
Conclusion
These test results support traditional medicinal use of, especially, aqueous extracts for the treatment of conditions such as diarrhea, and gonorrhea. The brine shrimp results depict the general trend among plants of the genus Terminalia, which are known to contain cytotoxic compounds such as hydrolysable tannins. These results warrant follow-up through bioassay-directed isolation of the active principles.
doi:10.1186/1472-6882-7-9
PMCID: PMC1851717  PMID: 17394672
18.  Antibacterial activity of some selected medicinal plants of Pakistan 
Background
Screening of the ethnobotenical plants is a pre-requisite to evaluate their therapeutic potential and it can lead to the isolation of new bioactive compounds.
Methods
The crude extracts and fractions of six medicinal important plants (Arisaema flavum, Debregeasia salicifolia, Carissa opaca, Pistacia integerrima, Aesculus indica, and Toona ciliata) were tested against three Gram positive and two Gram negative ATCC bacterial species using the agar well diffusion method.
Results
The crude extract of P. integerrima and A. indica were active against all tested bacterial strains (12-23 mm zone of inhibition). Other four plant's crude extracts (Arisaema flavum, Debregeasia salicifolia, Carissa opaca, and Toona ciliata) were active against different bacterial strains. The crude extracts showed varying level of bactericidal activity. The aqueous fractions of A. indica and P. integerrima crude extract showed maximum activity (19.66 and 16 mm, respectively) against B. subtilis, while the chloroform fractions of T. ciliata and D. salicifolia presented good antibacterial activities (13-17 mm zone of inhibition) against all the bacterial cultures tested.
Conclusion
The methanol fraction of Pistacia integerrima, chloroform fractions of Debregeasia salicifolia &Toona ciliata and aqueous fraction of Aesculus indica are suitable candidates for the development of novel antibacterial compounds.
doi:10.1186/1472-6882-11-52
PMCID: PMC3141602  PMID: 21718504
19.  Antimicrobial activity and cytotoxicity of triterpenes isolated from leaves of Maytenus undata (Celastraceae) 
Background
Plants of the genus Maytenus belong to the family Celastraceae and are widely used in folk medicine as anti-tumour, anti-asthmatic, analgesic, anti-inflammatory, antimicrobial and anti-ulcer agents, and as a treatment for stomach problems. The aim of this study was to isolate and identify active compounds with antifungal activity from Maytenus undata after a preliminary study highlighted promising activity in crude extracts.
Methods
Sequential extracts of M. undata leaves prepared using hexane, dichloromethane (DCM), acetone and methanol (MeOH) were tested for activity against Cryptococcus neoformans, a fungal organism implicated in opportunistic infections. Bioassay-guided fractionation of the hexane extract using C. neoformans as test organism was carried out to isolate antifungal compounds. The cytotoxicity of compounds isolated in sufficient quantities was evaluated using a tetrazolium-based colorimetric cellular assay (MTT) and a haemagglutination assay (HA).
Results
The hexane extract was most active with an MIC of 20 μg/ml against C. neoformans. The triterpene compounds friedelin (1), epifriedelanol (2), taraxerol (3), 3-oxo-11α-methoxyolean-12-ene-30-oic acid (4), 3-oxo-11α-hydroxyolean-12-ene-30-oic acid (5) and 3,11-dihydroxyolean-12-ene-30-oic acid (6) were isolated. Compound 6 was isolated for the first time from a plant species. The antimicrobial activity of compounds 1, 3, 5 and 6 was determined against a range of bacteria and fungi implicated in opportunistic and nosocomial infections. Compounds 5 and 6 were the most active against all the tested microorganisms with MIC values ranging between 24 and 63 μg/ml, except against Staphylococcus aureus which was relatively resistant. Compounds 1 and 3 had a low toxicity with an LC50 > 200 μg/ml towards Vero cells in the MTT assay. Compounds 5 and 6 were toxic with LC50 values of 6.03±0.02 and 2.98±0.01 μg/ml, respectively. Compounds 1 and 3 similarly were not toxic to the red blood cells (RBCs) but compounds 5 and 6 were toxic, showing HA titer values of 1.33 and 0.67 respectively.
Conclusions
Compounds 5 and 6 were the most active but were also relatively cytotoxic to monkey kidney cells and red blood cells, while the other isolated compounds were less active and less cytotoxic.
doi:10.1186/1472-6882-13-111
PMCID: PMC3711988  PMID: 23688235
Maytenus undata; Celastraceae; Antibacterial; Antifungal; Cytotoxicity; Haemagglutination assay
20.  Phytochemical analysis, antimicrobial and radical-scavenging properties of Acalypha manniana leaves 
SpringerPlus  2013;2:503.
Acalypha manniana (Euphorbiaceae) is a plant popularly used in Cameroon and in several parts of Africa for the treatment of various microbial diseases like diarrhea and skin infections.
The present study was designed to evaluate the phytochemical composition, antimicrobial and radical-scavenging activities of A. manniana methanol leaf extract and its fractions.
The methanol extract was partitioned into hexane, ethyl acetate and residual fractions and phytochemical analysis was conducted using standard methods. The broth microdilution method was used to evaluate the antimicrobial activity against nine bacterial species and four dermatophyte species. The free radical scavenging activities of the methanol extract and its fractions were evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay.
The results obtained showed that A. manniana contains alkaloids, tannins, anthocyanins, flavonoids, phenols and steroids. The methanol extract as well as the hexane, ethyl acetate and residual fractions exhibited both antibacterial and antidermatophytic activities that varied between the microbial species (MIC = 0.12 – 2.04 mg/mL). These tested samples also showed high radical-scavenging activities (RaS50 = 3.34 – 4.80 μg/mL) when compared with vitamin C used as reference antioxidant (RaS50 = 1.74 μg/mL).
These findings provide evidence that the studied plant possesses antimicrobial and antioxidant properties and may act as potential antioxidant for biological systems susceptible to free radical-mediated reactions.
doi:10.1186/2193-1801-2-503
PMCID: PMC3795206  PMID: 24130962
Acalypha manniana; Euphorbiaceae; Extracts; Phytochemicals; Antimicrobials; Free radical scavenging
21.  Cytotoxic effect of Alpinia scabra (Blume) Náves extracts on human breast and ovarian cancer cells 
Background
Alpinia scabra, locally known as 'Lengkuas raya’, is an aromatic, perennial and rhizomatous herb from the family Zingiberaceae. It is a wild species which grows largely on mountains at moderate elevations in Peninsular Malaysia, but it can also survive in the lowlands like in the states of Terengganu and Northern Johor. The present study reports the cytotoxic potential of A. scabra extracts from different parts of the plant.
Methods
The experimental approach in the present study was based on a bioassay-guided fractionation. The crude methanol and fractionated extracts (hexane, chloroform and water) from different parts of A. scabra (leaves, rhizomes, roots and pseudo stems) were prepared prior to the cytotoxicity evaluation against human ovarian (SKOV-3) and hormone-dependent breast (MCF7) carcinoma cells. The identified cytotoxic extracts were then subjected to chemical investigations in order to identify the active ingredients. A normal human lung fibroblast cell line (MRC-5) was used to determine the specificity for cancerous cells. The cytotoxic extracts and fractions were also subjected to morphological assessment, DNA fragmentation analysis and DAPI nuclear staining.
Results
The leaf (hexane and chloroform) and rhizome (chloroform) extracts showed high inhibitory effect against the tested cells. Ten fractions (LC1-LC10) were yielded after purification of the leaf chloroform extract. Fraction LC4 which showed excellent cytotoxic activity was further purified and resulted in 17 sub-fractions (VLC1-VLC17). Sub-fraction VLC9 showed excellent cytotoxicity against MCF7 and SKOV-3 cells but not toxic against normal MRC-5 cells. Meanwhile, eighteen fractions (RC1-RC18) were obtained after purification of the rhizome chloroform extract, of which fraction RC5 showed cytotoxicity against SKOV-3 cells with high selectivity index. There were marked morphological changes when observed using phase-contrast inverted microscope, DAPI nuclear staining and also DNA fragmentations in MCF7 and SKOV-3 cells after treatment with the cytotoxic extracts and fractions which were indicative of cell apoptosis. Methyl palmitate and methyl stearate were identified in the hexane leaf extract by GC-MS analysis.
Conclusions
The data obtained from the current study demonstrated that the cell death induced by cytotoxic extracts and fractions of A. scabra may be due to apoptosis induction which was characterized by apoptotic morphological changes and DNA fragmentation. The active ingredients in the leaf sub-fraction VLC9 and rhizome fraction RC5 may lead to valuable compounds that have the ability to kill cancer cells but not normal cells.
doi:10.1186/1472-6882-13-314
PMCID: PMC3833637  PMID: 24215354
Zingiberaceae; Alpinia scabra; Cytotoxic activity; Ovarian cancer; Breast cancer
22.  Antibacterial Activity of a Cardanol from Thai Apis mellifera Propolis 
Background: Propolis is a sticky, dark brown resinous residue made by bees that is derived from plant resins. It is used to construct and repair the nest, and in addition possesses several diverse bioactivities. Here, propolis from Apis mellifera from Nan province, Thailand, was tested for antibacterial activity against Gram+ve (Staphylococcus aureus and Paenibacillus larvae) and Gram-ve (Escherichia coli) bacteria.
Materials and methods: The three bacterial isolates were confirmed for species designation by Gram staining and analysis of the partial sequence of 16S rDNA. Propolis was sequentially extracted by methanol, dichloromethane and hexane. The antibacterial activity was determined by agar well diffusion and microbroth dilution assays using streptomycin as a positive control. The most active crude extract was further purified by quick column and adsorption chromatography. The apparent purity of each bioactive fraction was tested by thin layer chromatography. The chemical structure of the isolated bioactive compound was analyzed by nuclear magnetic resonance (NMR).
Results: Crude methanol extract of propolis showed the best antibacterial activity with a minimum inhibition concentration (MIC) value of 5 mg/mL for S. aureus and E. coli and 6.25 mg/mL for P. larvae. After quick column chromatography, only three active fractions were inhibitory to the growth of S. aureus and E. coli with MIC values of 6.25 and 31.3 µg/mL, respectively. Further adsorption chromatography yielded one pure bioactive fraction (A1A) with an IC50 value of 0.175 µg/mL for E. coli and 0.683 µg/mL for P. larvae, and was determined to be cardanol by NMR analysis. Scanning and transmission electron microscopy analysis revealed unusual shaped (especially in dividing cells), damaged and dead cells in cardanol-treated E. coli.
Conclusion: Thai propolis contains a promising antibacterial agent.
doi:10.7150/ijms.7373
PMCID: PMC3936026  PMID: 24578609
Antibacterial activity; Apis mellifera; Cardanol; Propolis; Nan province; Pathogen.
23.  Detection of Antimicrobial Compounds by Bioautography of Different Extracts of Leaves of Selected South African Tree Species 
The hexane, acetone, dichloromethane and methanol extracts of Combretum vendae A.E. van Wyk (Combretaceae), Commiphora harveyi (Engl.) Engl. (Burseraceae), Khaya anthotheca (Welm.) C.DC (Meliaceae), Kirkia wilmsii Engl. (Kirkiaceae), Loxostylis alata A. Spreng. ex Rchb. (Anacardiaceae), Ochna natalitia (Meisn.) Walp. (Ochnaceae) and Protorhus longifolia (Bernh. Ex C. Krauss) Engl. (Anacardiaceae) were screened for their antimicrobial activity. The test organisms included bacteria (Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus), and fungi (Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Microsporum canis and Sporothrix schenckii). A simple bioautographic procedure, involving spraying suspensions of the bacteria or fungi on thin layer chromatography (TLC) plates developed in solvents of varying polarities was used to detect the number of antibacterial and antifungal compounds present in the extracts. All the extracts had antimicrobial activity against at least one of the test microorganisms. This activity was denoted by white spots against a red-purple background on the TLC plates after spraying with tetrazolium violet. Twenty seven TLC plates; 9 for each solvent system and 3 different solvent systems per organism were tested in the bioautographic procedure. Of the bacteria tested, S. aureus was inhibited by the most compounds separated on the TLC plates from all the tested plants. Similarly, growth of the fungus C. neoformans was also inhibited by many compounds present in the extracts. Loxostylis alata appeared to be the plant extract with the highest number of inhibition bands when compared with other plants tested against both bacteria and fungi. This species was selected for in depth further study.
PMCID: PMC3005382  PMID: 21304615
Bioautography; Medicinal plants; Antifungal; Antibacterial; Synergism
24.  Iron (FeII) Chelation, Ferric Reducing Antioxidant Power, and Immune Modulating Potential of Arisaema jacquemontii (Himalayan Cobra Lily) 
BioMed Research International  2014;2014:179865.
This study explored the antioxidant and immunomodulatory potential of ethnomedicinally valuable species, namely, Arisaema jacquemontii of north-western Himalayan region. The tubers, leaves, and fruits of this plant were subjected to extraction using different solvents. In vitro antioxidant studies were performed in terms of chelation power on ferrous ions and FRAP assay. The crude methanol extract of leaves was found to harbour better chelating capacity (58% at 100 μg/mL) and reducing power (FRAP value 1085.4 ± 0.11 μMFe3+/g dry wt.) than all the other extracts. The crude methanol extract was thus further partitioned with solvents to yield five fractions. Antioxidant study of fractions suggested that the methanol fraction possessed significant chelation capacity (49.7% at 100 μg/mL) and reducing power with FRAP value of 1435.4 μM/g dry wt. The fractions were also studied for immune modulating potential where it was observed that hexane fraction had significant suppressive effect on mitogen induced T-cell and B-cell proliferation and remarkable stimulating effect on humoral response by 141% and on DTH response by 168% in immune suppressed mice as compared to the controls. Therefore, it can be concluded that A. jacquemontii leaves hold considerable antioxidant and immunomodulating potential and they can be explored further for the identification of their chemical composition for a better understanding of their biological activities.
doi:10.1155/2014/179865
PMCID: PMC4033394  PMID: 24895548
25.  Estimation of flavoniods, antimicrobial, antitumor and anticancer activity of Carissa opaca fruits 
Background
Carissa opaca Stapf ex Hanes fruits is traditionally used in the treatment of asthma, hepatitis and microbial infections. The present study was arranged to investigate the antimicrobial, cytotoxic and antitumor activity of various fractions of C. opaca extract and its bioactive metabolites responsible for that activity.
Methods
To characterize various fractions of C.opaca antibacterial, antifungal, cytotoxic and antitumor assays are used. Eight strains of bacteria including Bacillus subtilis, Enterobactor aerogenes, Escherichia coli, Klebsiella pneumoniae, Micrococcus luteus, Pseudomonas aeroginosa, Salmonella typhy, and Staphylococcus aureus and four strains of fungal viz: Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Fusarium solani are used. Brine shrimps and potato dics are used for anticancer and antitumor potency of extract. High performance liquid chromatography (HPLC) is utilized for determination of bioactive metabolites responsible for the activity.
Results
HPLC chromatogram revealed the presence of orientin, isoquercetin, myricetin and apigenin. Various fractions of C. oapca showed significant antibacterial, antitumor and anticancer activity. In case of C. opaca fruit inhibition growth of Aspergillus niger was ranged between 23.2 ± 1.36% to 43.3 ± 2.39%, Aspergillus flavus ranged between 27.6 ± 1.39% to 65.6 ± 3.44%, Aspergillus fumigatus ranged between 13.2 ± 1.00% to 52.4 ± 1.54% and Fusarium solani ranged between 10.5 ± 1.02% to 14.6 ± 1.74%.
Conclusion
It can be concluded that, various fractions of C.opaca are accessible source of ethno pharmacy as they are consumed in different areas of Pakistan with ultimate health compensations.
doi:10.1186/1472-6882-13-372
PMCID: PMC3893608  PMID: 24373124
Carissa opaca; High performance liquid chromatography; Escherichia coli; Anticancer

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