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1.  Antimicrobial, Antioxidant, and Wound Healing Properties of Kigelia africana (Lam.) Beneth. and Strophanthus hispidus DC. 
Microbial infections of various types of wounds are a challenge to the treatment of wounds and wound healing. The study was to investigate antimicrobial and antioxidant properties of methanol leaf and stem bark extracts of Kigelia africana and methanol leaf and root extracts of Strophanthus hispidus and also to determine wound healing properties of the extracts. The antimicrobial activities of the methanol extracts were determined against two Gram-positive and two Gram-negative bacteria and a fungus using agar diffusion and micro-dilution methods. The antioxidant activity was determined using 1,1-diphenyl-2-picryl–hydrazyl (DPPH) method. The influence of the extracts on rate of wound closure was investigated using the excision wound model and histopathological investigation of treated and untreated wound tissues performed. The MICs of leaf extract of K. africana against test organisms were 2.5–7.5 mg/mL and stem bark extract were 2.25–7.5 mg/mL. The leaf extract of S. hispidus had MIC range of 2.5–7.5 mg/mL and 2.5–10 mg/mL for root extract. The IC50 of leaf and stem bark extracts of K. africana were 56.9 and 13.7 μg/mL, respectively and leaf and root of S. hispidus were 49.8 and 45.1 μg/mL, respectively. K. africana extracts (7.5% w/w) showed significant (P < 0.05) wound contraction at day 7 with 72% of wound closure whiles significant (P < 0.05) wound contractions were observed on day 11 for stem bark of K. africana, leaf and root extracts of S. hispidus. Wound tissues treated with the extracts showed improved collagenation, re-epitheliazition and rapid granulation formation compared with untreated wound tissues. The extracts were found to contain alkaloids, saponins, tannins, flavonoids, carbohydrates, and sapogenetic glycosides. The HPLC finger-printing of the extracts were developed. The leaf, stem bark and root extracts of K. africana and S. hispidus exhibited antimicrobial, antioxidant, and enhanced wound healing properties and these may justify the medicinal uses of the plants for treatment of microbial infections and wounds.
PMCID: PMC3639673  PMID: 23662099
2.  Antioxidant activities of ethanol extracts and fractions of Crescentia cujete leaves and stem bark and the involvement of phenolic compounds 
Antioxidant compounds like phenols and flavonoids scavenge free radicals and thus inhibit the oxidative mechanisms that lead to control degenerative and other diseases. The aim of this study was to investigate the antioxidant activity in vitro, total phenolic and flavonoid contents in ethanol extracts and fractions of Crescentia cujete leaves and stem bark.
Crescentia cujete leaves and bark crude ethanol extract (CEE) and their partitionates petroleum ether (PEF), chloroform (CHF), ethyl acetate (EAF) and aqueous (AQF) were firstly prepared. Different established testing methods, such as 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical, ferric reducing power (FRP), and total antioxidant capacity (TAC) assays were used to detect the antioxidant activity. Further, the total yield, total phenolic (TPC) and total flavonoid contents (TFC) of CEE and all the fractions were determined. Ethanol extracts of both leaves and stem bark were also subjected to preliminary phytochemical screening to detect the presence of secondary metabolites, using standard phytochemical methods (Thin layer chromatography and spray reagents).
Phytochemical screening of crude ethanol extract of both leaves and stem bark revealed the presence of steroids, flavonoids, saponins, tannins, glycosides and terpenoids. All the fractions and CEE of leaves and bark exhibited antioxidant activities, however, EAF of leaves showing the highest antioxidant activity based on the results of DPPH, FRP and TAC assay tests. The above fraction has shown the significant DPPH scavenging activity (IC50 = 8.78 μg/ml) when compared with standard ascorbic acid (IC50 =7.68 μg/ml). The TAC and FRP activities increased with increasing crude extract/fractions content. The TPC (371.23 ± 15.77 mg GAE/g extract) and TFC (144.64 ± 5.82 mg QE/g extract) of EAF of leaves were found significantly higher as compared to other solvent fractions for both leaves and bark. TPC were highly correlated with the antioxidant activity (R2 = 0.9268 and 0.8515 in DPPH test for leaves and bark, respectively).
The results of the study show that leaves of C. cujete possesses significant free radical scavenging properties compared with stem bark and a clear correlation exists between the antioxidant activity and phenolic content.
PMCID: PMC3937116  PMID: 24495381
Calabash tree; Oxidative stress; Crude extracts; Free radicals; Anti-aging
3.  Antioxidant, antibacterial, cytotoxic, and apoptotic activity of stem bark extracts of Cephalotaxus griffithii Hook. f 
Cephalotaxus spp. are known to possess various therapeutic potentials. Cephalotaxus griffithii, however, has not been evaluated for its biological potential. The reason may be the remoteness and inaccessibility of the habitat where it is distributed. The main aim of this study was to: (1) evaluate multiple biological potentials of stem bark of C. griffithii, and (2) identify solvent extract of stem bark of C. griffithii to find the one with the highest specific biological activity.
Dried powder of stem bark of C. griffithii was exhaustively extracted serially by soaking in petroleum ether, acetone and methanol to fractionate the chemical constituents into individual fractions or extracts. The extracts were tested for total phenolic and flavonoid content, antioxidant (DPPH radical scavenging, superoxide radical scavenging, and reducing power models), antibacterial (disc diffusion assay on six bacterial strains), cytotoxic (MTT assay on HeLa cells), and apoptotic activity (fluorescence microscopy, DNA fragmentation assay, and flow cytometry on HeLa cells).
Among the three extracts of stem bark of C. griffithii, the acetone extract contained the highest amount of total phenolics and flavonoids and showed maximum antioxidant, antibacterial, cytotoxic (IC50 of 35.5 ± 0.6 μg/ml; P < 0.05), and apoptotic (46.3 ± 3.6% sub-G0/G1 population; P < 0.05) activity, followed by the methanol and petroleum ether extracts. However, there was no significant difference observed in IC50 values (DPPH scavenging assay) of the acetone and methanol extracts and the positive control (ascorbic acid). In contrast, superoxide radical scavenging assay-based antioxidant activity (IC50) of the acetone and methanol extracts was significantly lower than the positive control (P < 0.05). Correlation analysis suggested that phenolic and flavonoid content present in stem bark of C. griffithii extracts was responsible for the high antioxidant, cytotoxic, and apoptotic activity (P < 0.05).
Stem bark of C. griffithii has multiple biological effects. These results call for further chemical characterization of acetone extract of stem bark of C. griffithii for specific bioactivity.
PMCID: PMC3364904  PMID: 22469333
Cephalotaxus griffithii; Polyphenol; Antioxidant; Antibacterial; Cytotoxicity; Apoptosis
4.  Evaluation of Antimicrobial and Wound Healing Potential of Justicia flava and Lannea welwitschii 
Microbial infections of various types of wounds are a challenge to the treatment of wounds and wound healing. The aim of the study is to determine the antimicrobial, antioxidant, and in vivo wound healing properties of methanol leaf extracts of Justicia flava and Lannea welwitschii. The antimicrobial activity was investigated using agar well diffusion and microdilution methods. The free radical scavenging activity of the methanol leaf extracts was performed using 1,1-diphenyl-2-picryl-hydrazyl (DPPH). The rate of wound contraction was determined using excision model. The test organisms used were Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 4853, Bacillus subtilis NTCC 10073, Staphylococcus aureus ATCC 25923, and clinical strains of Candida albicans. The MICs of methanol leaf extract of J. flava against test organisms were E. coli (7.5 mg/mL); P. aeruginosa (7.5 mg/mL); S. aureus (5 mg/mL); B. subtilis (7.5 mg/mL); and C. albicans (5 mg/mL). The MICs of methanol leaf extract of L. welwitschii against test organisms were E. coli (5 mg/mL); P. aeruginosa (10 mg/mL); S. aureus (5 mg/mL); B. subtilis (2.5 mg/mL); and C. albicans (2.5 mg/mL). The MBC/MFC of the extract was between 10 and 50 mg/mL. The IC50 of the reference antioxidant, α-tocopherol, was 1.5 μg/mL and the methanol leaf extracts of J. flava and L. welwitschii had IC50 of 65.3 μg/mL and 81.8 μg/mL, respectively. The methanol leaf extracts of J. flava and L. welwitschii gave a significant reduction in wound size as compared to the untreated. The rates of wound closure after the application of the extracts (7.5% w/w) were compared to the untreated wounds. On the 9th day, J. flava extract had a percentage wound closure of 99% (P < 0.01) and that of L. welwitschii exhibited wound closure of 95% (P < 0.05) on the 13th day compared to the untreated wounds. The two extracts significantly (P < 0.01) increased the tensile strength of wounds compared to the untreated wounds. The extracts treated wound tissues showed improved angiogenesis, collagenation, and reepithelialization compared to the untreated wound tissues. The preliminary phytochemical screening of J. flava and L. welwitschii leaf extracts revealed the presence of tannins, alkaloids, flavonoids, and glycosides. The above results indicate that methanol leaf extracts of J. flava and L. welwitschii possess antimicrobial and wound healing properties which may justify the traditional uses of J. flava and L. welwitschii in the treatment of wounds and infections.
PMCID: PMC3789403  PMID: 24159350
5.  Wound healing activities of different extracts of Centella asiatica in incision and burn wound models: an experimental animal study 
The efficacy of Centella asiatica for incision and burn wounds are not fully understood. Here, we report the wound healing activities of sequential hexane, ethyl acetate, methanol, and water extracts of Centella asiatica in incision and partial-thickness burn wound models in rats.
Male Sprague–Dawley rats weighing 250–300 g were randomly divided into incision and burn wound groups. Each group was stratified into seven subgroups: (1) untreated; (2) NSS-; (3) Tween 20®- (vehicle control); (4) hexane extract-; (5) ethyl acetate extract-; (6) methanol extract-; and (7) aqueous extract-treated groups. The test substances were applied topically once daily. The tensile strength of the incision wound was measured on the seventh day after wound infliction. The general appearance and degree of wound healing of the burn wound were assessed on Days 3, 7, 10 and 14 after burn injury and prior to histopathological evaluation.
On the seventh day after wound infliction, the tensile strength of incision wound in all extract-treated groups was significantly higher than that of the vehicle control (Tween 20®), but comparable to the NSS-treated group. The degrees of healing in the burn wound with the four extracts were significantly higher than that of the control on Days 3, 10 and 14. Histopathological findings on Day 14 after burn injury revealed prominent fibrinoid necrosis and incomplete epithelialization in the control and untreated groups, whereas fully developed epithelialization and keratinization were observed in all extract-treated groups. Analysis by thin layer chromatography demonstrated that the phyto-constituents β-sitosterol, asiatic acid, and asiaticoside and madecassocide were present in the hexane, ethyl acetate and methanol extracts, respectively.
All extracts of Centella asiatica facilitate the wound healing process in both incision and burn wounds. Asiatic acid in the ethyl acetate extract seemed to be the most active component for healing the wound.
PMCID: PMC3492213  PMID: 22817824
Centella asiatica; Wound healing; Incision wound; Burn wound; Asiatic acid; β-sitosterol; Asiaticoside; Madecassocide
6.  Antibacterial Activity of some Medicinal Mangroves against Antibiotic Resistant Pathogenic Bacteria 
The antibacterial activity of the leaves and bark of mangrove plants, Avicennia marina, A. officinalis, Bruguiera sexangula, Exoecaria agallocha, Lumnitzera racemosa, and Rhizophora apiculata was evaluated against antibiotic resistant pathogenic bacteria, Staphylococcus aureus and Proteus sp. Soxhlet extracts of petroleum ether, ethyl acetate, ethanol and water were prepared and evaluated the antibacterial activity using agar diffusion method. Most of the plant extracts showed promising antibacterial activity against both bacterial species. However, higher antibacterial activity was observed for Staphylococcus aureus than Proteus sp. The highest antibacterial activity was shown by ethyl acetate of mature leaf extracts of E. agallocha for Staphylococcus aureus. All ethyl acetate extracts showed higher inhibition against S. aureus while some extracts of chloroform, ethyl acetate and ethanol gave inhibition against Proteus sp. None of the petroleum ether and aqueous extracts showed inhibition against Proteus sp. All fresh plant materials did also show more antibacterial activity against both bacterial strains than did dried plant extracts. Antibacterial activity of fresh and dried plant materials reduced for both bacterial strains with time after extraction. Since L. racemosa and A. marina gave the best inhibition for bacterial species, they were used for further investigations. Charcoal treated plant extracts of L. racemosa and A. marina were able to inhibit both bacterial strains more than those of untreated plant extracts. Phytochemical screening of mature leaf, bark of L. racemosa and leaf extracts of A. marina has been carried out and revealed that leaf and bark contained alkaloids, steroids, triterpenoids and flavonoids. None of the above extracts indicate the presence of saponins and cardiac glycosides. Separated bands of extracts by TLC analysis showed antibacterial activity against S. aureus.
PMCID: PMC2929774  PMID: 20838519
Antibacterial activity; inhibition; mangroves; soxhlet extraction
7.  Evaluation of Antioxidant and Wound Healing Potentials of Sphaeranthus amaranthoides Burm.f. 
BioMed Research International  2013;2013:607109.
Background. Sphaeranthus amaranthoides commonly known as sivakaranthai is used in folklore medicine for the treatment of skin diseases. Methods. The antioxidant activity of the extract and its fraction was evaluated by using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, total antioxidant capacity, and total phenolic content. The tested plant extracts showed variable degrees of antioxidant activity. In the present study, methanolic extract of the whole plant of S. amaranthoides and a flavonoid fraction obtained from column chromatography were studied for wound healing activity by incorporating the sample in simple ointment base. Wound healing activity was studied in excision wound model in rats, following which, wound contraction, period of epithelization, hydroxyproline content, and collagen levels in the scab were studied. Results. Methanolic extract showed the highest antioxidant effect (72.05%) and diethyl ether extract has the least (29.34%) compared to the standard (74.53%). Treatment of wound with ointment containing 5% (w/w) methanolic extract and 5% (w/w) flavonoid fraction exhibited better wound healing activity than positive control (silver sulfadiazine). Finally, histopathology studies conformed wound healing activity in Sphaeranthus amaranthoides. The methanolic extract and flavonoid fraction exhibited good wound healing activity probably due to the presence of phenolic and flavonoid constituents. The methanolic extract and flavonoid fraction significantly enhanced the rate of wound contraction and the period of epithelialization comparable to silver sulfadiazine.
PMCID: PMC3581292  PMID: 23509751
8.  Wound Healing Potential of Methanol Extract of Spathodea Campanulata Stem Bark Formulated into a Topical Preparation 
This study evaluated the wound healing potential of Spathodea campanulata stem bark in Sprague Dawley rats using the excision wound model. The methanol extract contained glycosides, flavonoids and tannins, and was relatively stable when stored at the room temperature for six (6) months. Solvent-free, semi-solid extract of S. campanulata was incorporated into an aqueous cream and applied (10 % w/w and 20 % w/w) on excision wounds of thirty two (32) rats. Cicatrin® cream was used as a standard wound healing agent. Prior to the remedial cream application, done later on twice daily, sixteen (16) rats had their wounds infected with Staphylococcus aureus, while in the remaining sixteen the wounds were kept clean. The surface area of the excision wounds was monitored planimetrically every four (4) days until a complete wound closure or healing took place. Excision wounds treated with 20 % w/w Spathodea cream and Cicatrin® cream showed a rapid and comparable decrease (p > 0.05) in wound size. In uninfected wounds, both 20 % w/w Spathodea cream and Cicatrin® cream application resulted in ∼ 95 %-wound closure seen on Day 20, and a complete closure seen on Day 24. In infected wounds, both 20 % w/w Spathodea cream and Cicatrin® cream administration led to ∼ 91 %-wound closure on Day 24 and a complete wound contraction on Day 28. The results of this study justify the folkloric use of S. campanulata stem bark to the effect of wound treatment.
PMCID: PMC3252224  PMID: 22468000
Spathodea campanulata; wound healing; excision wound model; Spathodea cream; Cicatrin® cream; wound contraction
9.  Studies on Wound Healing Activity of Heliotropium indicum Linn. Leaves on Rats 
ISRN Pharmacology  2011;2011:847980.
The petroleum ether, chloroform, methanol, and aqueous extracts of Heliotropium indicum Linn. (Family: Boraginaceae) were separately evaluated for their wound healing activity in rats using excision (normal and infected), incision, and dead space wound models. The effects of test samples on the rate of wound healing were assessed by the rate of wound closure, period of epithelialisation, wound breaking strength, weights of the granulation tissue, determination of hydroxyproline, super oxide dismutase (SOD), catalase, and histopathology of the granulation tissues. Nitrofurazone (0.2% w/w) in simple ointment I. P. was used as reference standard for the activity comparison. The results revealed significant promotion of wound healing with both methanol and aqueous extracts with more promising activity with the methanol extract compared to other extracts under study. In the wound infection model (with S. aureus and P. aeruginosa), the methanol extract showed significant healing activity similar to the reference standard nitrofurazone. Significant increase in the granulation tissue weight, increased hydroxyproline content, and increased activity of SOD and catalase level with the animals treated with methanol extract in dead space wound model further augmented the wound healing potential of H. indicum. The present work substantiates its validity of the folklore use.
PMCID: PMC3198611  PMID: 22084720
10.  Investigation of Wound Healing Activity of Methanolic Extract of Stem Bark of Mimusops Elengi Linn 
The present study was aimed to evaluate the wound healing activity of extract of bark part of Mimusops elengi. It is well-known plant in Indian traditional medicines. On the basis of traditional use and literature references, this plant was selected for wound healing potential. A methanolic extract of bark parts of Mimusops elengi was examined for wound healing activity in the form of ointment in three types of wound models on mice: the excision, the incision and dead space wound model. The extract ointments showed considerable response in all the above said wound models as comparable to those of a standard drug Betadine ointment in terms of wound contracting ability, wound closure time, tensile strength and dry granuloma weight. Histological analysis was also consistent with the proposal that Mimusops elengi bark extract exhibits significant wound healing
PMCID: PMC3252694  PMID: 22238490
Mimusops elengi; Wound healing; Betadine; methanolic extract
11.  Potentials of leaves of Aspilia africana (Compositae) in wound care: an experimental evaluation 
The potentials of the leaves of the haemorrhage plant, Aspilia africana C. D Adams (Compositae) in wound care was evaluated using experimental models. A. africana, which is widespread in Africa, is used in traditional medicine to stop bleeding from wounds, clean the surfaces of sores, in the treatment of rheumatic pains, bee and scorpion stings and for removal of opacities and foreign bodies from the eyes. The present study was undertaken to evaluate the potentials for use of leaves of this plant in wound care.
The effect of the methanol extract (ME) and the hexane (HF) and methanol (MF) fractions (obtained by cold maceration and graded solvent extraction respectively) on bleeding/clotting time of fresh experimentally-induced wounds in rats, coagulation time of whole rat blood, growth of microbial wound contaminants and rate of healing of experimentally-induced wounds in rats were studied as well as the acute toxicity and lethality (LD50) of the methanol extract and phytochemical analysis of the extract and fractions.
The extract and fractions significantly (P < 0.05) reduced bleeding/clotting time in rats and decreased coagulation time of whole rat blood in order of magnitude of effect: MF>ME>HF. Also, the extract and fractions caused varying degrees of inhibition of the growth of clinical isolates of Pseudomonas fluorescens and Staphylococcus aureus, as well as typed strains of Ps. aeruginosa (ATCC 10145) and Staph. aureus (ATCC 12600), and reduced epithelialisation period of wounds experimentally-induced in rats. Acute toxicity and lethality (LD50) test in mice established an i.p LD50 of 894 mg/kg for the methanol extract (ME). Phytochemical analysis revealed the presence of alkaloids, saponins, tannins, flavonoids, resins, sterols, terpenoids and carbohydrates.
The leaves of A. africana possess constituents capable of arresting wound bleeding, inhibiting the growth of microbial wound contaminants and accelerating wound healing which suggest good potentials for use in wound care.
PMCID: PMC1940021  PMID: 17623087
12.  Blechnum Orientale Linn - a fern with potential as antioxidant, anticancer and antibacterial agent 
Blechnum orientale Linn. (Blechnaceae) is used ethnomedicinally for the treatment of various skin diseases, stomach pain, urinary bladder complaints and sterilization of women. The aim of the study was to evaluate antioxidant, anticancer and antibacterial activity of five solvent fractions obtained from the methanol extract of the leaves of Blechnum orientale Linn.
Five solvent fractions were obtained from the methanol extract of B. orientale through successive partitioning with petroleum ether, chloroform, ethyl acetate, butanol and water. Total phenolic content was assessed using Folin-Ciocalteu's method. The antioxidant activity was determined by measuring the scavenging activity of DPPH radicals. Cytotoxic activity was tested against four cancer cell lines and a non-malignant cell using MTT assay. Antibacterial activity was assessed using the disc diffusion and broth microdilution assays. Standard phytochemical screening tests for saponins, tannins, terpenoids, flavonoids and alkaloids were also conducted.
The ethyl acetate, butanol and water fractions possessed strong radical scavenging activity (IC50 8.6-13.0 μg/ml) and cytotoxic activity towards human colon cancer cell HT-29 (IC50 27.5-42.8 μg/ml). The three extracts were also effective against all Gram-positive bacteria tested: Bacillus cereus, Micrococcus luteus, methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA) and Stapylococcus epidermidis(minimum inhibitory concentration MIC 15.6-250 μg/ml; minimum bactericidal concentration MBC 15.6-250 μg/ml). Phytochemical analysis revealed the presence of flavonoids, terpenoids and tannins. Ethyl acetate and butanol fractions showed highest total phenolic content (675-804 mg gallic acid equivalent/g).
The results indicate that this fern is a potential candidate to be used as an antioxidant agent, for colon cancer therapy and for treatment of MRSA infections and other MSSA/Gram-positive bacterial infectious diseases.
PMCID: PMC2881084  PMID: 20429956
13.  In vitro and in vivo antidermatophytic activity of the dichloromethane-methanol (1:1 v/v) extract from the stem bark of Polyscias fulva Hiern (Araliaceae) 
During the last decades, the number of people suffering from dermatophytoses has seriously increased, mainly due to the development of resistant strains of microorganisms to a range of formally efficient antibiotics. Polyscias fulva, a medium size tree which grows in the West Region of Cameroon is traditionally used for local application against dermatoses and orally against venereal infections. The dichloromethane-methanol (1:1 v/v) extract from the stem bark of Polyscias fulva was evaluated for its in vitro and in vivo antifungal activities.
The plant extract was prepared by maceration of its stem bark powder in CH2Cl2-MeOH (1:1 v/v). The extract obtained was successively partitioned in hexane, ethyl acetate and n-butanol. Phytochemical screening was performed using standard methods. In vitro antidermatophytic activity was assayed by the well diffusion and broth microdilution methods. The degree of dermal irritation of the crude extract was determined in guinea pigs using the occluded dermal irritation test method. The in vivo antidermatophytic activity of the extract-oil formulation (1.25, 2.5 and 5% w/w concentrations) was evaluated using Trichophyton mentagrophytes-induced dermatophytosis in a guinea pigs model.
Phytochemical screening indicated that, the crude extract, ethyl acetate, n-butanol and residue fractions contain in general saponins, tannins, alkaloids, anthraquinones and phenols while the hexane fraction contains only alkaloids. The ethyl-acetate, n-butanol and residue fractions displayed higher antifungal activities (MIC = 0.125-0.5 mg.mL-1) against eight dermatophytes as compared to the crude extract (MIC = 0.5-1 mg.mL-1). This latter appeared to have slight perceptible erythema effects on guinea pigs as the primary irritation index (PII) was calculated to be 0.54. In vivo, the antidermatophytic activities of the extract-oil formulations were dose-dependent. Griseofulvin-oil 5% at 0.01 g/kg and formulated extract-oil (5%) at 0.1 g/kg eradicated the microbial infection after thirteen and fourteen days of daily treatment respectively.
The results of preclinical in vitro and in vivo evaluations indicate that the extract-oil formulation at 5% may constitute an alternative means to alleviate fungal infections caused by dermatophytes.
PMCID: PMC3658906  PMID: 23641972
Antidermatophytic activity; Polyscias fulva; Extract-oil formulation; Irritation test; Primary irritation index; Guinea pigs
14.  Antioxidant and anti-dermatophytic properties leaf and stem bark of Xylosma longifolium clos 
The present study was carried out to assess the phytochemical and anti-dermatophytic effect of the leaf and bark extracts of Xylosma longifolium Clos. The leaf and stem bark are used by the indigenous people of Manipur, India for treatment of skin diseases.
The leaves and stem barks of Xylosma longifolium were extracted using petroleum ether, chloroform and methanol respectively. The different extracts of each plant parts were tested for antioxidant activity using DPPH assay. The phenolic content was assayed using Folin-Ciocalteu colorimetric method. Each extracts was further analysed by RP-HPLC to quantify some individual flavonoid components. The anti-dermatophytic activity was evaluated both by agar diffusion method and micro wells dilution method against the Microsporum boullardii MTCC 6059, M. canis (MTCC 2820 and MTCC 32700), M. gypseum MTCC 2819, Trichophyton ajelloi MTCC 4878, T. rubrum (MTCC 296 and MTCC 3272).
The free radical scavenging activity values were ranged from 0.7 to 1.41 mg/ml and 0.6 to 1.23 mg/ml, respectively for leaf and stem bark extracts. The amount of total phenolic contents of the extracts occurred in both leaf and bark in the range of 12 to 56.6 mg GAE/100 g and 16 to 58 mg GAE/100 g respectively. RP-HPLC analysis for flavonoids revealed the presence of two major flavonoid compounds, rutin and catechin. Kaempferol was in trace or absent. Methanol leaf extract showed significant low inhibitory effect against tested fungus Trichophyton ajelloi MTCC 4878 (0.140625 mg/ml) as the most sensitive. These finding suggest that the methanol leaf extract tested contain compounds with antimicrobial properties.
The results of our study may partially justify the folkloric uses on the plant studied and further provide an evidence that the leaf extract of Xylosma longifolium might be indeed a potential sources of antimicrobial agents.
PMCID: PMC3718714  PMID: 23819459
Xylosma Longifolium; Phenolic; Flavonoid; Antioxidant; RP-HPLC; Anti-Dermatophytic
15.  Evaluation of the Wound Healing Potential of Achillea biebersteinii Afan. (Asteraceae) by In Vivo Excision and Incision Models 
Achillea species are widely used for diarrhea, abdominal pain, stomachache and healing of wounds in folk medicine. To evaluate the wound healing activity of the plant, extracts were prepared with different solvents; hexane, chloroform, ethyl acetate and methanol, respectively from the roots of Achillea biebersteinii. Linear incision by using tensiometer and circular excision wound models were employed on mice and rats. The wound healing effect was comparatively evaluated with the standard skin ointment Madecassol. The n-hexane extract treated groups of animals showed 84.2% contraction, which was close to contraction value of the reference drug Madecassol (100%). On the other hand the same extract on incision wound model demonstrated a significant increase (40.1%) in wound tensile strength as compared to other groups. The results of histoptological examination supported the outcome of linear incision and circular excision wound models as well. The experimental data demonstrated that A. biebersteinii displayed remarkable wound healing activity.
PMCID: PMC3136533  PMID: 19546149
16.  Wound Healing Activity of Rubus sanctus Schreber (Rosaceae): Preclinical Study in Animal Models 
Young shoots of Rubus species have been used for healing of wounds, infected insect bites and pimples in folk medicine for ages. In order to evaluate the wound healing activity of Rubus sanctus, four different extracts were prepared from the whole aerial parts of the plant by using n-hexane, chloroform, ethyl acetate and methanol, respectively. Incision wound healing model by using tensiometer on rats and excision model on mice were employed to assess the activity. Remarkable wound healing activity was observed with the ointment formulation of the methanol extract at 1% concentration on the mentioned models. The results of histopathological examination also supported the outcome of both incision and excision wound models. The wound healing effect was comparatively evaluated with a reference ointment Madecassol. The experimental data confirmed the ethnobotanical usage of R. sanctus.
PMCID: PMC3139958  PMID: 19755505
17.  In vitro Antimicrobial Activity and Phytochemical Screening of Clematis Species Indigenous to Ethiopia 
The leaves extracts of two indigenous plants of Ethiopia: Clematis longicauda steud ex A. Rich. and Clematis burgensis Engl. are used in Southwestern Ethiopia to treat otorrhoea and eczema. Antimicrobial activity and MIC of crude extracts were determined by disk diffusion and broth dilution. Phytochemical screening was performed on the extracts. The methanol and petroleum ether extracts of both plants showed antibacterial and antifungal activity. Sensitivity of reference strains was concentration dependent. Methanol and petroleum ether extracts of C. burgensis leaves exerted greater inhibitory effects than C. longicauda extracts whereas aqueous extracts of both plants were inactive. The MIC study revealed a concentration of 0.78 mg/ml on bacteria and 3.125 mg/ml on fungi for methanol extract and 1.56 mg/ml on both fungi and bacteria for petroleum ether extract. Phytochemical screening results indicated the presence of proteins, fixed oils, carbohydrates, tannins, saponins, flavonoids, and steroids. Preliminary chromatographic investigation showed fluorescing spots with Rf values that ranged from 0.05 to 0.96 for phenolic compounds and saponins. As the study is one of the first reports on the two indigenous species of Clematis; isolation, purification and characterization of the different primary and secondary metabolites may further yield alternative options to the microbial chemotherapy.
PMCID: PMC3507342  PMID: 23204619
Clematis longicauda; Clematis burgensis; phytochemical screening; antimicrobial activity; ethiopia
18.  Ixora coccinea Enhances Cutaneous Wound Healing by Upregulating the Expression of Collagen and Basic Fibroblast Growth Factor 
ISRN Pharmacology  2014;2014:751824.
Background. Ixora coccinea L. (Rubiaceae) has been documented for traditional use in hypertension, menstrual irregularities, sprain, chronic ulcer, and skin diseases. In the present study, I. coccinea was subjected to in vitro and in vivo wound healing investigation. Methods. Petroleum ether, chloroform, methanol, and water sequential I. coccinea leaves extracts were evaluated for in vitro antioxidant, antimicrobial, and fibroblast proliferation activities. The promising I. coccinea methanol extract (IxME) was screened for in vivo wound healing activity in Wistar rat using circular excision model. Wound contraction measurement, hydroxyproline quantification, and western blot for collagen type III (COL3A1), basic fibroblast growth factor (bFGF), and Smad-2, -3, -4, and -7 was performed with 7-day postoperative wound granulation tissue. Gentamicin sulfate (0.01% w/w) hydrogel was used as reference standard. Results. IxME showed the potent antimicrobial, antioxidant activities, with significant fibroblast proliferation inducing activity, as compared to all other extracts. In vivo study confirmed the wound healing accelerating potential of IxME, as evidenced by faster wound contraction, higher hydroxyproline content, and improved histopathology of granulation tissue. Western blot analysis revealed that the topical application of I. coccinea methanol extract stimulates the fibroblast growth factor and Smad mediated collagen production in wound tissue.
PMCID: PMC3928818  PMID: 24624303
Ancient Science of Life  2005;25(2):57-61.
The preliminary phytochemical analysis of Limonia acidissima plant parts such as bark, leaf, rind, pulp and seed showed the presence of alkaloids, flavonoids, steroids, saponins, glycosides, phenols, gum and mucilage, fixed oils and fats, resins and tannins. Among the five plant parts, pulp possessed high amount of protein and the carbohydrate content was more in seeds and rind is rich was amino acid. The methanolic extracts of L. acidissima plant parts were tested against Escherichia coli and Staphylococcus aureus using disc diffusion method. The extracts from different parts showed varying degrees of antimicrobial activity.
PMCID: PMC3330905  PMID: 22557192
Limonia acidissima; stomachic; astringent; diuretic; cardiotonic; carminative; bowel infections; antidote
20.  Antibacterial, antidiarrhoeal, and cytotoxic activities of methanol extract and its fractions of Caesalpinia bonducella (L.) Roxb leaves 
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.
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.
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.
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.
PMCID: PMC3661353  PMID: 23663985
Caesalpinia bonducella; Antimicrobial; Antidiarrhoeal; Cytotoxicity
21.  Euphorbia hirta accelerates fibroblast proliferation and Smad-mediated collagen production in rat excision wound 
Pharmacognosy Magazine  2014;10(Suppl 3):S534-S542.
Euphorbia hirta L. (Euphorbiaceae) is a traditional herbal medicine used for treatment of various diseases.
E. hirta was investigated for in vitro/in vivo wound healing activity using human dermal fibroblast cell line and Wistar rats.
Materials and Methods:
Petroleum ether, chloroform, methanol and water successive extracts of E. hirta leaves were evaluated for antioxidant, antimicrobial and fibroblast proliferation activities. Among different extracts, the promising methanol extract was screened for wound healing activity in Wistar rats, using gentamicin sulfate (0.01% w/w) as a reference. Wound contraction, hydroxyproline content and the protein expression of COL3A1, bFGF, Smad-2,-3,-4 and -7 were measured.
The E. hirta methanol extract showed a potent antimicrobial (MIC 0.250 mg/ml against Escherichia coli and Klebsiella pneumoniae, both), antioxidant activities (IC50 = 10.57 μg/ml, 2,2-diphenyl-1-picrylhydrazyl; 850.23 μg/ml, superoxide-anion radical scavenging activity and 23.63 mg gallic acid equivalent per gram extract) with significant fibroblast proliferating activity (112% at 12.5 μg/ml) as compared to other extracts. In vivo study also supported the wound healing potential of methanol extract, as evidenced by faster wound contraction, higher hydroxyproline (4.240 mg/100 mg tissue) and improved histopathology of granulation tissue as compared to control groups and gentamicin sulfate-treated ones. Western blot also revealed a significantly altered expression of Smad-mediated proteins resulting in collagen production.
The study suggested that E. hirta accelerates the wound healing by augmenting the fibroblast proliferation and Smad-mediated collagen production in wound tissue.
PMCID: PMC4189269  PMID: 25298671
Basic fibroblast growth factor; Euphorbia hirta; hydroxyproline; smad-mediated collagen production; wound healing
22.  In vitro and in vivo Antiinflammatory Activity of Clerodendrum paniculatum Linn. Leaves 
Preliminary phytochemical screening showed the presence of terpenes, flavonoids, tannins, alkaloids, phenolic acid, sterols, and glycosides. This study was intended to evaluate the antiinflammatory activity of various extracts of fresh leaves of Clerodendrum paniculatum Linn experimentally by in vitro (human red blood cell membrane stabilization method) and in vivo methods (0.1 ml of 1% w/v carrageenan-induced rat paw oedema model). Petroleum ether, chloroform, ethyl acetate, alcohol, and aqueous extracts were screened for in vitro antiinflammatory activity. Petroleum ether and chloroform extracts which showed, best in vitro antiinflammatory activity was screened for in vivo antiinflammatory activity at the dose level of 200 and 400 mg/kg. Indomethacin at the dose level of 10 mg/kg was used as reference standard drug. Both the extracts showed a dose dependent significant (P<0.001) reduction in paw edema when compared to the control, at all the time intervals and comparable to indomethacin (reference standard) treated group. The results of the present study demonstrate that petroleum ether and chloroform extracts possess significant (P<0.001) antiinflammatory potential which provide scientific basis for the traditional claims of Clerodendrum paniculatum Linn leaves as an antiinflammatory drug.
PMCID: PMC3783760  PMID: 24082358
Antiinflammatory activity; carrageenan; Clerodendrum paniculatum Linn; human red blood cells membrane; verbenaceae
23.  In vitro antibacterial activity of Tabernaemontana alternifolia (Roxb) stem bark aqueous extracts against clinical isolates of methicillin resistant Staphylococcus aureus 
The rise of antibiotic resistance among methicillin resistant Staphylococcus aureus (MRSA), have caused concerns for the treatment of MRSA infections. Hence, search for an alternative therapy for these infections is inevitable. Folk Indian medicine refers to the use of leaf and stem bark powder of Tabernaemontana alternifolia (Roxb) in treatment of skin infections, but no scientific report establishes its antibacterial activity.
Direct aqueous extracts and sequential aqueous extracts of the stem bark of T. alternifolia (using petroleum ether and ethyl acetate as other solvents) were prepared by soxhlet extraction. The antibiotic sensitivity profiles of the clinical isolates were determined against 18 antibiotics using disc diffusion method. The isolates were identified by 16S rRNA gene sequencing. The methicillin resistance among S. aureus (MRSA) was confirmed by PCR amplification of mecA gene. The disc diffusion method was used to determine the antibacterial activity of the extracts. The micro-dilution method was used to determine the minimum inhibitory concentration (MIC) of the extract against the test organism. To further evaluate the therapeutic potential of the extract, cell cytotoxicity was checked on Vero cells by MTT assay. Chemical profiling of the extract was done by HPTLC method.
The aqueous extracts of T. alternifolia stem bark exhibited antibacterial activity against Gram-positive microorganisms, particularly against clinical isolates of MRSA and vancomycin resistant S. aureus (VRSA). The minimum inhibitory concentration (MIC) of extract against the isolates ranged from 600–800 μg/ml. The extract did not exhibit cytotoxic activity against Vero cells even at the concentration of 4 mg/ml. The chemical profiling revealed presence of alkaloids, flavonoids, coumarins, saponins and steroids. Petroleum ether and ethyl acetate extracts did not exhibit antibacterial activity.
Our results offer a scientific basis for the traditional use of T. alternifolia in the treatment of skin infections, showing that the plant extract has an enormous potential as a prospective alternative therapy against MRSA skin infections. The present study lays the basis for future studies, to validate the possible use of T. alternifolia as a candidate in the treatment of MRSA infections.
PMCID: PMC3851168  PMID: 24066905
Tabernaemontana alternifolia (Roxb); Anti-MRSA; Cytotoxicity; Plant extract; Antimicrobial
24.  Antidiarrheal activity of extracts and compound from Trilepisium madagascariense stem bark 
Indian Journal of Pharmacology  2010;42(3):157-163.
The present study was performed to evaluate the preventive and curative antidiarrheal effects of the methanol extract, fractions and compound from the stem bark of Trilepisium madagascariense in rats.
Materials and Methods:
The methanol extract from the stem bark of T. madagascariense, its fractions (n-hexane, ethyl acetate, n-butanol and aqueous residue) and compound (obtained from further column chromatography of the ethyl acetate fraction) were evaluated for the antidiarrheal activity in rats. These test samples (at 100, 200 and 400 mg/kg for the extract and fractions and 2.5 mg/kg for compound) were assayed on the latent periods, purging indices and fecal frequencies in castor oil-induced diarrhea. Gastrointestinal transit and castor oil-induced enteropooling assays were conducted. Shigella-induced diarrhea was assayed. Blood chemistry and fecal Shigella load were examined.
The fractionation of the ethyl acetate fraction from the methanol extract of T. madagascariense afforded a known compound [isoliquiritigenin (1)]. Compound 1 increased the latent period of diarrhea induction (179.40 min) compared to the saline control (60.80 min). The purging indices, fecal frequencies and intestinal enteropooling decreased with an increase in the dose of test samples. The blood cell counts, sera creatinine and fecal Shigella load decreased significantly (P ≤ 0.05) in the plant extract-treated rats compared to the saline control.
The results of our study, being reported for the first time, provide clear evidence that the methanol extract, fractions and isoliquiritigenin from T. madagascariense stem bark possess antidiarrheal activities.
PMCID: PMC2937317  PMID: 20871767
Antidiarrheal; castor oil; isoliquiritigenin; Shigella; Trilepisium madagascariense
25.  Effects of Payena dasyphylla (Miq.) on hyaluronidase enzyme activity and metalloproteinases protein expressions in interleukin-1β stimulated human chondrocytes cells 
Hyaluronidases have been found as the target enzymes in the development of osteoarthritis (OA) disease. While there is still no curative treatment for this disease, recent studies on the treatment of OA were focused on the effectiveness of natural products which are expected to improve the symptoms with minimal side effects. The aim of this study was to screen selected Malaysian plants on their anti-hyaluronidase activity as well as to evaluate the active plant and its derived fractions on its potential anti-arthritic and antioxidant activities.
A total of 20 methanolic crude extracts (bark and leaf) from ten different plants were screened using a colorimetric hyaluronidase enzymatic assay. The active plant extract (Payena dasyphylla) was then studied for its hyaluronidase inhibitory activity in the interleukin-1β (IL-1β) stimulated human chondrocytes cell line (NHAC-kn) using zymography method. The Payena dasyphylla methanolic bark extract was then fractionated into several fractions in where the ethyl acetate (EA) fraction was evaluated for its inhibitory effects on the HYAL1 and HYAL2 gene expressions using reverse transcription-polymerase chain reaction (RT-PCR) technique. While the MMP-3 and MMP-13 protein expressions were evaluated using western blot method. The phenolic and flavonoid contents of the three fractions as well as the antioxidant property of the EA fraction were also evaluated.
Bark extract of Payena dasyphylla (100 μg/ml) showed the highest inhibitory activity against bovine testicular hyaluronidase with 91.63%. The plant extract also inhibited hyaluronidase expression in the cultured human chondrocyte cells in response to IL-1β (100 ng/ml). Similarly, treatment with Payena dasyphylla ethyl acetate (EA) fraction (100 μg/ml) inhibited the HYAL1 and HYAL2 mRNA gene expressions as well as MMP-3 and MMP-13 protein expression in a dose dependent manner. Payena dasyphylla EA fraction has demonstrated the highest amount of phenolic and flavonoid content with 168.62 ± 10.93 mg GAE/g and 95.96 ± 2.96 mg RE/g respectively as compared to water and hexane fractions. In addition, the Payena dasyphylla EA fraction showed strong antioxidant activity with IC50 value of 11.64 ± 1.69 μg/mL.
These findings have shown that Payena dasyphylla might contained potential phenolic compounds that inhibiting the key enzyme in osteoarthritis development, which is the hyaluronidase enzyme through interruption of HYAL1 and HYAL1 gene expressions. The degradation of cartilage could also be inhibited by the plant through suppression of MMP-3 and MMP-13 protein expressions. We also reported that the inhibitory effect of Payena dasyphylla on hyaluronidase activity and expression might be due to its anti-oxidant property.
PMCID: PMC3848702  PMID: 23971790
Osteoarthritis; Hyaluronidase; MMP-3; MMP-13; Payena dasyphylla

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