Shining insights: Deciphering the biogenic synthesis of Ajuga bracteosa-mediated gold nanoparticles with advanced microscopy techniques.

In this study, gold nanoparticles (AuNPs) were bioreduced from Ajuga bracteosa, a medicinal herb known for its therapeutic properties against various diseases. Different fractions of the plant extract were used, including the methanolic fraction (ABMF), the n-hexane fraction (ABHF), the chloroform fraction (ABCF), and the aqueous extract for AuNPs synthesis. The characterization of AuNPs was performed using UV-Vis spectrophotometry, FT-IR, XRD, EDX, and TEM. UV-Vis spectroscopy confirmed the formation of AuNPs, with peaks observed at 555 nm. FT-IR analysis indicated strong capping of phytochemicals on the surface of AuNPs, which was supported by higher total phenolic contents (TPC) and total flavonoid contents (TFC) in AuNPs. XRD results showed high crystallinity and a smaller size distribution of AuNPs. TEM analysis revealed the spherical shape of AuNPs, with an average size of 29 ± 10 nm. The biologically synthesized AuNPs exhibited superior antibacterial, antioxidant, and cytotoxic activities compared to the plant extract fractions. The presence of active biomolecules in A. bracteosa, such as neoclerodan flavonol glycosides, diterpenoids, phytoecdysone, and iridoid glycosides, contributed to the enhanced biological activities of AuNPs. Overall, this research highlights the potential of A. bracteosa-derived AuNPs for various biomedical applications due to their remarkable therapeutic properties and effective capping by phytochemicals. RESEARCH HIGHLIGHTS: This research underscores the growing significance of herbal medicine in contemporary healthcare by exploring the therapeutic potential of Ajuga bracteosa and gold nanoparticles (AuNPs). The study highlights the notable efficacy of A. bracteosa leaf extracts and AuNPs in treating bacterial infections, demonstrating their bactericidal effects on a range of strains. The anti-inflammatory properties of plant extracts and nanoparticles are evidenced through paw edema method suggesting their applicability in managing inflammatory conditions. These findings position A. bracteosa and AuNPs as potential candidates for alternative and effective approaches to modern medication.

Comparative analysis of chloroplast genome and new insights into phylogenetic relationships of Ajuga and common adulterants.

Introduction: The potential contamination of herbal medicinal products poses a significant concern for consumer health. Given the limited availability of genetic information concerning Ajuga species, it becomes imperative to incorporate supplementary molecular markers to enhance and ensure accurate species identification. Methods: In this study, the chloroplast (cp) genomes of seven species of the genus Ajuag were sequenced, de novo assembled and characterized. Results: exhibiting lengths ranging from 150,342 bp to 150,472 bp, encompassing 86 - 88 protein-coding genes (PCGs), 35 - 37 transfer RNA, and eight ribosomal RNA. The repetitive sequences, codon uses, and cp genomes of seven species were highly conserved, and PCGs were the reliable molecular markers for investigating the phylogenetic relationship within the Ajuga genus. Moreover, four mutation hotspot regions (accD-psaI, atpH-atpI, ndhC-trnV(UAC), and ndhF-rpl23) were identified within cp genomes of Ajuga, which could help distinguish A. bracteosa and its contaminants. Based on cp genomes and PCGs, the phylogenetic tree preliminary confirmed the position of Ajuga within the Lamiaceae family. It strongly supported a sister relationship between Subsect. Genevense and Subsect. Biflorae, suggesting the merger of Subsect. Biflorae and Subsect. Genevenses into one group rather than maintaining separate categorizations. Additionally, molecular clock analysis estimated the divergence time of Ajuga to be around 7.78 million years ago. Discussion: The species authentication, phylogeny, and evolution analyses of the Ajuga species may benefit from the above findings.

Biofabrication of Fe3O4 Nanoparticles from Spirogyra hyalina and Ajuga bracteosa and Their Antibacterial Applications.

Iron oxide nanoparticles (NPs) have attracted substantial interest due to their superparamagnetic features, biocompatibility, and nontoxicity. The latest progress in the biological production of Fe3O4 NPs by green methods has improved their quality and biological applications significantly. In this study, the fabrication of iron oxide NPs from Spirogyra hyalina and Ajuga bracteosa was conducted via an easy, environmentally friendly, and cost-effective process. The fabricated Fe3O4 NPs were characterized using various analytical methods to study their unique properties. UV-Vis absorption peaks were observed in algal and plant-based Fe3O4 NPs at 289 nm and 306 nm, respectively. Fourier transform infrared (FTIR) spectroscopy analyzed diverse bioactive phytochemicals present in algal and plant extracts that functioned as stabilizing and capping agents in the fabrication of algal and plant-based Fe3O4 NPs. X-ray diffraction of NPs revealed the crystalline nature of both biofabricated Fe3O4 NPs and their small size. Scanning electron microscopy (SEM) revealed that algae and plant-based Fe3O4 NPs are spherical and rod-shaped, averaging 52 nm and 75 nm in size. Energy dispersive X-ray spectroscopy showed that the green-synthesized Fe3O4 NPs require a high mass percentage of iron and oxygen to ensure their synthesis. The fabricated plant-based Fe3O4 NPs exhibited stronger antioxidant properties than algal-based Fe3O4 NPs. The algal-based NPs showed efficient antibacterial potential against E. coli, while the plant-based Fe3O4 NPs displayed a higher zone of inhibition against S. aureus. Moreover, plant-based Fe3O4 NPs exhibited superior scavenging and antibacterial potential compared to the algal-based Fe3O4 NPs. This might be due to the greater number of phytochemicals in plants that surround the NPs during their green fabrication. Hence, the capping of bioactive agents over iron oxide NPs improves antibacterial applications.

A comparative study to evaluate the effects of antibiotics, plant extracts and fluoride-based toothpaste on the oral pathogens isolated from patients with gum diseases in Pakistan / Um estudo comparativo para avaliar os efeitos de antibióticos, extratos vegetais e creme dental à base de flúor sobre os patógenos orais isolados de pacientes com doenças gengivais no Paquistão

Oral diseases caused by various microorganisms are common around the world. Scientific research has now been focusing on novel medicines to overcome bacterial resistance and antibiotics side effects; therefore, the current study was designed to assess the efficacy of certain antibiotics, toothpaste, and medicinal plant extracts (Ajuga bracteosa and Curcuma longa) versus the bacterial pathogens isolated from the human oral cavity. A total of 130 samples were collected from Khyber Teaching Hospital Peshawar, Pakistan, among those 27 species isolated, and eight bacterial species were identified from the samples. Among all the bacterial species, Staphylococcus aureus (29.62%) and Proteus mirabilis (22.2%) were found to be more prevalent oral pathogens. In comparison, the least pervasive microbes were Proteus vulgaris, Shigella sonnei, Escherichia coli and Aeromonas hydrophila. The study also suggested that dental problems were more prevalent in males (41-50 years of age) than females. Among the eight antibiotics used in the study, the most promising results were shown by Foxicillin against A. hydrophila. The survey of TP1 revealed that it showed more potent antagonist activity against Proteus vulgaris as compared TP2 and TP3 that might be due to the high content of fluoride. The Curcuma longa showed more significant activity than Ajuga bracteosa (Stem, leaves and root) extracts. The data obtained through this study revealed that antibiotics were more effective for oral bacterial pathogens than toothpaste and plant extracts which showed moderate and low activity, respectively. Therefore, it is suggested that the active compounds in individual medicinal plants like Curcuma longa and Ajuga bracteosa could replace the antibiotics when used in daily routine as tooth cleansers or mouth rinses.

As doenças bucais causadas por vários microrganismos são comuns em todo o mundo. A pesquisa científica agora tem se concentrado em novos medicamentos para superar a resistência bacteriana e os efeitos colaterais dos antibióticos; portanto, o presente estudo foi desenhado para avaliar a eficácia de certos antibióticos, pasta de dente e extratos de plantas medicinais (Ajuga bracteosa e Curcuma longa) contra os patógenos bacterianos isolados da cavidade oral humana. No total, 130 amostras foram coletadas do Khyber Teaching Hospital Peshawar, Paquistão, entre essas, 27 espécies foram isoladas e oito espécies bacterianas foram identificadas a partir das amostras. Entre todas as espécies bacterianas, Staphylococcus aureus (29.62%) e Proteus mirabilis (22.2%) foram os patógenos orais mais prevalentes. Em comparação, os micróbios menos difundidos foram Proteus vulgaris, Shigella sonnei, Escherichia coli e Aeromonas hydrophila. O estudo também sugeriu que os problemas dentários eram mais prevalentes em homens (41-50 anos de idade) do que em mulheres. Entre os oito antibióticos usados no estudo, os resultados mais promissores foram mostrados pelo Foxicillin contra A. hydrophila. A pesquisa de TP1 revelou que ele mostrou atividade antagonista mais potente contra Proteus vulgaris em comparação a TP2 e TP3, o que pode ser devido ao alto teor de flúor. A Curcuma longa apresentou atividade mais significativa em relação aos extratos de Ajuga bracteosa (caule, folhas e raiz). Os dados obtidos neste estudo revelaram que os antibióticos foram mais eficazes para os patógenos bacterianos orais do que os dentifrícios e os extratos vegetais que apresentaram atividade moderada e baixa, respectivamente. Portanto, sugere-se que os compostos ativos em plantas medicinais individuais como Curcuma longa e Ajuga bracteosa possam substituir os antibióticos quando usados na rotina diária como limpadores de dentes ou enxaguatórios bucais.

A comparative study to evaluate the effects of antibiotics, plant extracts and fluoride-based toothpaste on the oral pathogens isolated from patients with gum diseases in Pakistan

Abstract Oral diseases caused by various microorganisms are common around the world. Scientific research has now been focusing on novel medicines to overcome bacterial resistance and antibiotics side effects; therefore, the current study was designed to assess the efficacy of certain antibiotics, toothpaste, and medicinal plant extracts (Ajuga bracteosa and Curcuma longa) versus the bacterial pathogens isolated from the human oral cavity. A total of 130 samples were collected from Khyber Teaching Hospital Peshawar, Pakistan, among those 27 species isolated, and eight bacterial species were identified from the samples. Among all the bacterial species, Staphylococcus aureus (29.62%) and Proteus mirabilis (22.2%) were found to be more prevalent oral pathogens. In comparison, the least pervasive microbes were Proteus vulgaris, Shigella sonnei, Escherichia coli and Aeromonas hydrophila. The study also suggested that dental problems were more prevalent in males (41-50 years of age) than females. Among the eight antibiotics used in the study, the most promising results were shown by Foxicillin against A. hydrophila. The survey of TP1 revealed that it showed more potent antagonist activity against Proteus vulgaris as compared TP2 and TP3 that might be due to the high content of fluoride. The Curcuma longa showed more significant activity than Ajuga bracteosa (Stem, leaves and root) extracts. The data obtained through this study revealed that antibiotics were more effective for oral bacterial pathogens than toothpaste and plant extracts which showed moderate and low activity, respectively. Therefore, it is suggested that the active compounds in individual medicinal plants like Curcuma longa and Ajuga bracteosa could replace the antibiotics when used in daily routine as tooth cleansers or mouth rinses.

Resumo As doenças bucais causadas por vários microrganismos são comuns em todo o mundo. A pesquisa científica agora tem se concentrado em novos medicamentos para superar a resistência bacteriana e os efeitos colaterais dos antibióticos; portanto, o presente estudo foi desenhado para avaliar a eficácia de certos antibióticos, pasta de dente e extratos de plantas medicinais (Ajuga bracteosa e Curcuma longa) contra os patógenos bacterianos isolados da cavidade oral humana. No total, 130 amostras foram coletadas do Khyber Teaching Hospital Peshawar, Paquistão, entre essas, 27 espécies foram isoladas e oito espécies bacterianas foram identificadas a partir das amostras. Entre todas as espécies bacterianas, Staphylococcus aureus (29.62%) e Proteus mirabilis (22.2%) foram os patógenos orais mais prevalentes. Em comparação, os micróbios menos difundidos foram Proteus vulgaris, Shigella sonnei, Escherichia coli e Aeromonas hydrophila. O estudo também sugeriu que os problemas dentários eram mais prevalentes em homens (41-50 anos de idade) do que em mulheres. Entre os oito antibióticos usados no estudo, os resultados mais promissores foram mostrados pelo Foxicillin contra A. hydrophila. A pesquisa de TP1 revelou que ele mostrou atividade antagonista mais potente contra Proteus vulgaris em comparação a TP2 e TP3, o que pode ser devido ao alto teor de flúor. A Curcuma longa apresentou atividade mais significativa em relação aos extratos de Ajuga bracteosa (caule, folhas e raiz). Os dados obtidos neste estudo revelaram que os antibióticos foram mais eficazes para os patógenos bacterianos orais do que os dentifrícios e os extratos vegetais que apresentaram atividade moderada e baixa, respectivamente. Portanto, sugere-se que os compostos ativos em plantas medicinais individuais como Curcuma longa e Ajuga bracteosa possam substituir os antibióticos quando usados na rotina diária como limpadores de dentes ou enxaguatórios bucais.

A comparative study to evaluate the effects of antibiotics, plant extracts and fluoride-based toothpaste on the oral pathogens isolated from patients with gum diseases in Pakistan / Um estudo comparativo para avaliar os efeitos de antibióticos, extratos vegetais e creme dental à base de flúor sobre os patógenos orais isolados de pacientes com doenças gengivais no Paquistão

Abstract Oral diseases caused by various microorganisms are common around the world. Scientific research has now been focusing on novel medicines to overcome bacterial resistance and antibiotics side effects; therefore, the current study was designed to assess the efficacy of certain antibiotics, toothpaste, and medicinal plant extracts (Ajuga bracteosa and Curcuma longa) versus the bacterial pathogens isolated from the human oral cavity. A total of 130 samples were collected from Khyber Teaching Hospital Peshawar, Pakistan, among those 27 species isolated, and eight bacterial species were identified from the samples. Among all the bacterial species, Staphylococcus aureus (29.62%) and Proteus mirabilis (22.2%) were found to be more prevalent oral pathogens. In comparison, the least pervasive microbes were Proteus vulgaris, Shigella sonnei, Escherichia coli and Aeromonas hydrophila. The study also suggested that dental problems were more prevalent in males (41-50 years of age) than females. Among the eight antibiotics used in the study, the most promising results were shown by Foxicillin against A. hydrophila. The survey of TP1 revealed that it showed more potent antagonist activity against Proteus vulgaris as compared TP2 and TP3 that might be due to the high content of fluoride. The Curcuma longa showed more significant activity than Ajuga bracteosa (Stem, leaves and root) extracts. The data obtained through this study revealed that antibiotics were more effective for oral bacterial pathogens than toothpaste and plant extracts which showed moderate and low activity, respectively. Therefore, it is suggested that the active compounds in individual medicinal plants like Curcuma longa and Ajuga bracteosa could replace the antibiotics when used in daily routine as tooth cleansers or mouth rinses.

Resumo As doenças bucais causadas por vários microrganismos são comuns em todo o mundo. A pesquisa científica agora tem se concentrado em novos medicamentos para superar a resistência bacteriana e os efeitos colaterais dos antibióticos; portanto, o presente estudo foi desenhado para avaliar a eficácia de certos antibióticos, pasta de dente e extratos de plantas medicinais (Ajuga bracteosa e Curcuma longa) contra os patógenos bacterianos isolados da cavidade oral humana. No total, 130 amostras foram coletadas do Khyber Teaching Hospital Peshawar, Paquistão, entre essas, 27 espécies foram isoladas e oito espécies bacterianas foram identificadas a partir das amostras. Entre todas as espécies bacterianas, Staphylococcus aureus (29.62%) e Proteus mirabilis (22.2%) foram os patógenos orais mais prevalentes. Em comparação, os micróbios menos difundidos foram Proteus vulgaris, Shigella sonnei, Escherichia coli e Aeromonas hydrophila. O estudo também sugeriu que os problemas dentários eram mais prevalentes em homens (41-50 anos de idade) do que em mulheres. Entre os oito antibióticos usados ​​no estudo, os resultados mais promissores foram mostrados pelo Foxicillin contra A. hydrophila. A pesquisa de TP1 revelou que ele mostrou atividade antagonista mais potente contra Proteus vulgaris em comparação a TP2 e TP3, o que pode ser devido ao alto teor de flúor. A Curcuma longa apresentou atividade mais significativa em relação aos extratos de Ajuga bracteosa (caule, folhas e raiz). Os dados obtidos neste estudo revelaram que os antibióticos foram mais eficazes para os patógenos bacterianos orais do que os dentifrícios e os extratos vegetais que apresentaram atividade moderada e baixa, respectivamente. Portanto, sugere-se que os compostos ativos em plantas medicinais individuais como Curcuma longa e Ajuga bracteosa possam substituir os antibióticos quando usados ​​na rotina diária como limpadores de dentes ou enxaguatórios bucais.

Cytotoxicity, Anti-diabetic, and Hepato-protective Potential of Ajuga bracteosa-conjugated Silver Nanoparticles in Balb/c Mice.

BACKGROUND: Ajuga bracteosa is a traditional herb used against various diseases. OBJECTIVES: Current research aimed to investigate the anti-diabetic and hepato-protective effect of green synthesized silver nanoparticles (ABAgNPs) using Ajuga bracteosa aqueous extract (ABaqu). METHODS: In vitro anti-diabetic and cytotoxic effects were carried out via α- glucosidase inhibition, brine shrimp lethality, and protein kinase inhibition assays. For in vivo screening of 200 mg/kg and 400 mg/kg of both ABAgNPs and ABaqu in alloxan-induced and CCl4-induced Swiss albino mice were used. Liver and kidney functional markers, hematology, and histopathological studies were carried out after 14 days of administration. RESULTS: In vivo antidiabetic and anti-cancerous effects showed valuable anti-hyperglycemic and hepatoprotective potential when mice were treated with ABaqu and ABAgNPs. A significant reduction in the blood glucose level was recorded when ABaqu and ABAgNPs were administrated orally compared to Glibenclamide treated group. Significant reduction in ALT, AST, ALP, urea, uric acid, and creatinine was recorded in ABaqu and ABAgNPs treated diabetic mice. The hepato-protective findings indicated that ALT, ALP, AST were elevated in CCl4-induced mice while declined in both ABAgNPs and ABaqu treated CCl4-induced mice. Histopathological examination revealed that ABAgNPs have hepato-protective activity. CONCLUSION: It was concluded that ABAgNPs and ABaqu possessed strong anti-diabetic and hepatoprotective phytoconstituents, which could be used in the prevention of diseases.

Antibacterial, Antihemolytic, Cytotoxic, Anticancer, and Antileishmanial Effects of Ajuga bracteosa Transgenic Plants.

Herbal and traditional medicines can play a pivotal role in combating cancer and neglected tropical diseases. Ajuga bracteosa, family Lamiaceae, is an important medicinal plant. The genetic transformation of A. bracteosa with rol genes of Agrobacterium rhizogenes further enhances its metabolic content. This study aimed at undertaking the molecular, phytochemical, and in vitro biological analysis of A. bracteosa extracts. We transformed the A. bracteosa plant with rol genes and raised the regenerants from the hairy roots. Transgenic integration and expression of rolB were confirmed by conventional polymerase chain reaction (PCR) and qPCR analysis. The methanol: chloroform crude extracts of wild-type plants and transgenic regenerants were screened for in vitro antibacterial, antihemolytic, cytotoxic, anticancer, and leishmanial activity. Among all plants, transgenic line 3 (ABRL3) showed the highest expression of the rolB gene. Fourier transform infra-red (FTIR) analysis confirmed the enhanced number of functional groups of active compounds in all transgenic lines. Moreover, ABRL3 exhibited the highest antibacterial activity, minimum hemolytic activity (CC50 = 7293.05 ± 7 µg/mL) and maximum antileishmanial activity (IC50 of 56.16 ± 2 µg/mL). ABRL1 demonstrated the most prominent brine shrimp cytotoxicity (LD5039.6 ± 4 µg/mL). ABRL3 was most effective against various human cancer cell lines with an IC50 of 57.1 ± 2.2 µg/mL, 46.2 ± 1.1 µg/mL, 72.4 ± 1.3 µg/mL, 73.3 ± 2.1 µg/mL, 98.7 ± 1.6 µg/mL, and 97.1 ± 2.5 µg/mL against HepG2, LM3, A549, HT29, MCF-7, and MDA-MB-231, respectively. Overall, these transgenic extracts may offer a cheaper therapeutic source than the more expensive synthetic drugs.

Polyphenol Rich Ajuga bracteosa Transgenic Regenerants Display Better Pharmacological Potential.

Ajuga bracteosa Wall. ex Benth. is an endangered medicinal herb traditionally used against different ailments. The present study aimed to create new insight into the fundamental mechanisms of genetic transformation and the biological activities of this plant. We transformed the A. bracteosa plant with rol genes of Agrobacterium rhizogenes and raised the regenerants from the hairy roots. These transgenic regenerants were screened for in vitro antioxidant activities, a range of in vivo assays, elemental analysis, polyphenol content, and different phytochemicals found through HPLC. Among 18 polyphenolic standards, kaempferol was most abundant in all transgenic lines. Furthermore, transgenic line 3 (ABRL3) showed maximum phenolics and flavonoids content among all tested plant extracts. ABRL3 also demonstrated the highest total antioxidant capacity (8.16 ± 1 µg AAE/mg), total reducing power, (6.60 ± 1.17 µg AAE/mg), DPPH activity (IC50 = 59.5 ± 0.8 µg/mL), hydroxyl ion scavenging (IC50 = 122.5 ± 0.90 µg/mL), and iron-chelating power (IC50 = 154.8 ± 2 µg/mL). Moreover, transformed plant extracts produced significant analgesic, anti-inflammatory, anticoagulant, and antidepressant activities in BALB/c mice models. In conclusion, transgenic regenerants of A. bracteosa pose better antioxidant and pharmacological properties under the effect of rol genes as compared to wild-type plants.

In vitro examination of anti-parasitic, anti-Alzheimer, insecticidal and cytotoxic potential of Ajuga bracteosa Wallich leaves extracts.

This research study is mainly focused to evaluate the anti-parasitic, insecticidal, cytotoxic and anti-alzheimer potential of various leaf extracts of Ajuga bracteosa Wallich ex Bentham. 04 different extracts were prepared using solvent of different polarity to determine the best candidate for potent bioactivity i.e. n-hexane (NH), Ethyl acetate (EA), Ethanol (EL) and Chloroform (CH). Concentrations of each extracts were made specified for all activities. All extracts were exploited for broad range of biomedical applications including leishmaniasis, in vitro anti-Alzheimer, insecticidal and cytotoxic studies. Our results showed that A. bracteosa n-hexane extract was highly active against Leishmania Tropica with significant inhibition of 58 ± 1.61 for promastigote and 63 ± 2.29 for amastigote at 1000 µg/mL. Furthermore, promising anti-alzheimer activity acetylcholinesterase (AChE) 46 ± 0.83 and butrylcholineterase (BChE) 49 ± 1.17 was noted for n-hexane. The insecticidal potential of these extracts were test against five different insects (Rhyzopertha dominica, Trogoderma granarium, Tribolium castaneum, Sitophilus oryze, and Callosobruchus analis). The higest mortality rate of insecticidal activity was recorded by n-hexane followed by Ethyl acetate whereas ethanol extract was found to be less effective against all the test species. Significant cytotoxic potential of each plant sample against Artemia salina thus aware us for further detailed research to find out novel drugs. Based on our results we believe that Ajuga bracteosa could be used to develop as a potential botanical insecticide against different insect and pests, such as aphids as well as an excellent source for the compound isolation as anti-tumor agent.

Synergistic Antibacterial Efficacy of Biogenic Synthesized Silver Nanoparticles using Ajuga bractosa with Standard Antibiotics: A Study Against Bacterial Pathogens.

BACKGROUND: Multi-drug resistance in bacterial pathogens is a major concern of today. Green synthesis technology is being used to cure infectious diseases. OBJECTIVES: The aim of the current research was to analyze the antibacterial, antioxidant, and phytochemical screening of green synthesized silver nanoparticles using Ajuga bracteosa. METHODS: Extract of A. bracteosa was prepared by maceration technique. Silver nanoparticles were synthesized using A. bracteosa extract and were confirmed by UV-Vis spectrophotometer, Scanning Electron Microscope (SEM) and Fourier Transform Infrared Spectroscopy (FTIR). The antibacterial, anti-biofilm, cell proliferation inhibition, TLC-Bioautography, TLC-Spot screening, antioxidant, and phytochemical screening were also investigated. RESULTS: UV-Vis spectrum and Scanning electron microscopy confirmed the synthesis of green nanoparticles at 400 nm with tube-like structures. FTIR spectrum showed that functional groups of nanoparticles have a role in capping and stability of AgNP. Agar well diffusion assay represented the maximum antibacterial effect of ABAgNPs against Escherichia coli, Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, and Pseudomonas aeruginosa at 0.10 g/mL concentration compared to ABaqu. Two types of interactions among nanoparticles, aqueous extract, and antibiotics (Synergistic and additive) were recorded against tested pathogens. Crystal violet, MTT, TLC-bio-autography, and spot screening supported the findings of the antibacterial assay. Highest antioxidant potential effect in ABaqu was 14.62% (DPPH) and 13.64% (ABTS) while 4.85% (DPPH) and 4.86% (ABTS) was recorded in ABAgNPs. Presence of phytochemical constituents showed pharmacological importance. CONCLUSION: It was concluded that green synthesis is an innovative technology in which natural products are conjugated with metallic particles and are used against infectious pathogens. The current research showed the significant use of green nanoparticles against etiological agents.

Photo-dependent somatic embryogenesis from non-embryogenic calli and its polyphenolics content in high-valued medicinal plant of Ajuga bracteosa.

Ajuga bracteosa (A. bracteosa) is one of the critically endangered and high-valued medicinal plants worldwide. Light is one of the major factor or stimulus involved in the morphogenic responses and bioactive compounds production in various medicinal plants. In this study, unique properties of colored lights have been observed on induction of somatic embryos from non-embryonic calli cultures of A. bracteosa. The maximum callogenic response (92.32%) from leaf explants was observed on Murashige and Skoog (MS) medium augmented with benzyl adenine (BA; 2.0 l-1) and 2, 4-Dichlorophenoxy acetic acid (2.4-D; 1.0 mg l-1). Calli cultures with same hormonal concentrations were placed under different spectral lights for somatic embryogenesis and photochemical variations. Red lights were found effective for maximum somatic embryos induction (92.75%) with optimum biomass accumulation (152.64 g l-1) on day 40. Similarly, among all the spectral lights, red light exhibited the highest DPPH-radical scavenging activity (DRSA; 92.86%). In contrast, blue lights induced maximum biosynthesis of chemically important total phenolics content and total flavonoids content (TPC; 0.264 and TFC; 0.06 mg/g-DW), respectively. Furthermore, blue, green and red lights also enhanced phenolics and production, polyphenolics content and total polyphenolics production in somatic embryos. It is concluded that exposure of calli cultures to colored lights provides an effective and promising in vitro technique for conservation of endangered A. bracteosa species and enhancement of its bioactive compounds. Steps should be taken to adopt these strategies/ techniques at a larger scale in order to yield maximum benefits from this highly valued medicinal plant species.

Antimutagenic activity of compounds isolated from Ajuga bracteosa Wall ex. Benth against EMS induced mutagenicity in mice.

Ajuga bracteosa Wall ex. Benth. (Lamiaceae) has been reported to possess many biological activities including antibacterial, antifungal, antispasmodic and antioxidant activity but there is no report as such on its mutagenic and/or anti-mutagenic activity. The aim of the present study was to isolate compounds from the methanol extract of the aerial parts of Ajuga bracteosa and determine their anti-mutagenic activity against the mutagen, EMS in animal model mice. The study was undertaken in order to corroborate the traditional use of the plant Ajuga bracteosa. The compounds were isolated from the methanol extract of the aerial parts of Ajuga bracteosa using silica gel column chromatography. Structural elucidation of the isolated compounds was done using spectral data analysis and comparison with literature. High performance liquid chromatography (HPLC) was used for the qualitative and quantitative determination of the isolated compounds in the crude methanol extract. The isolated compounds and standard drug were evaluated in vivo for antimutagenic activity against EMS induced mutagenicity taking mice as model organism by micronucleus and chromosomal aberration tests. Four major compounds were identified as 1) 14, 15-dihydroajugapitin 2) ß- Sitosterol 3) Stigmasterol and 4) 8-O-acetylharpagide. A quick and sensitive HPLC method was developed for qualitative and quantitative determination of three isolated marker compounds from Ajuga bracteosa. 14, 15-dihydroajugapitin reduced the micronuclei by 85.10%, followed by ß- Sitosterol (72.3%) while as 8-O-acetylharpagide reduced the micronuclei by 46%. It is therefore evident from the present study that the plant contains rich source of anticancer and antimutagenic drugs.

Polarity based characterization of biologically active extracts of Ajuga bracteosa Wall. ex Benth. and RP-HPLC analysis.

BACKGROUND: The concept of botanical therapeutics has revitalized due to wide importance of plant derived pharmaceuticals. Therefore, the ameliorative characteristics of Ajuga bracteosa were studied. METHODS: Total phenolic content, flavonoid content, antioxidant capacity, reducing power and free-radical scavenging activity were determined colorimetrically. Specific polyphenols were quantified by RP-HPLC analysis. Preliminary cytotoxicity was tested using brine shrimp lethality assay while antiproliferative activity against THP-1 and Hep-G2 cell lines was determined by MTT and SRB protocols respectively. Antileishmanial potential was assessed via MTT colorimetric method. To investigate antidiabetic prospect, α-amylase inhibition assay was adopted whereas disc diffusion method was used to detect likely protein kinase inhibitory, antibacterial and antifungal activities. RESULTS: Among fifteen different extracts, maximum total phenolic content (10.75 ± 0.70 µg GAE/mg DW), total reducing power (23.90 ± 0.70 µg AAE/mg DW) and total antioxidant capacity (11.30 ± 0.80 µg AAE/mg DW) were exhibited by methanol extract with superlative percent extract recovery (17.50 ± 0.80% w/w). Chloroform-methanol extract demonstrated maximum flavonoid content (4.10 ± 0.40 µg QE/mg DW) and ethanol extract exhibited greatest radical scavenging activity (IC50 14.40 ± 0.20 µg/ml). RP-HPLC based quantification confirmed polyphenols such as pyrocatechol, gallic acid, resorcinol, catechin, chlorogenic acid, caffeic acid, syringic acid, p-coumaric acid, ferulic acid, vanillic acid, coumarin, sinapinic acid, trans-cinnamic acid, rutin, quercetin and kaempferol. The brine shrimp lethality assay ranked 78.60% extracts as cytotoxic (LC50 ≤ 250 µg/ml) whereas significant THP-1 inhibition was shown by methanol-acetone extract (IC50 4.70 ± 0.43 µg/ml). The antiproliferative activity against Hep-G2 hepatoma cancer cell line was demonstrated by n-hexane, ethylacetate and methanol-distilled water (IC50 8.65-8.95 µg/ml) extracts. Methanol extract displayed prominent protein kinase inhibitory activity (MIC 12.5 µg/disc) while n-hexane extract revealed remarkable antileishmanial activity (IC50 4.69 ± 0.01 µg/ml). The antidiabetic potential was confirmed by n-hexane extract (44.70 ± 0.30% α-amylase inhibition at 200 µg/ml concentration) while a moderate antibacterial and antifungal activities were unveiled. CONCLUSION: The variation in biological spectrum resulted due to use of multiple solvent systems for extraction. We also deduce that the valuable information gathered can be utilized for discovery of anticancer, antileishmanial, antioxidant and antidiabetic bioactive lead candidates.

Induction of Mitotic Delay in Pharyngeal and Nasopharyngeal Carcinoma Cells Using an Aqueous Extract of Ajuga bracteosa.

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, for which radiotherapy and/or chemotherapy are the primary treatment methods. Many herbs are known to have potential uses in chemotherapy; however, the mechanisms underlying the observed antitumor activity of Ajuga bracteosa (AB) against NPC remain unclear. We explored the antitumor effects of AB, which was shown specifically to induce mitotic delay in pharyngeal (Detroit 562) and nasopharyngeal (Hone-1) cancer cells. Proliferation of cancer cells after exposure to aqueous extract of A. bracteosa (AEAB) was assessed using the MTT assay. DNA content and cell cycle arrest induction were analyzed using flow cytometry. The expression of checkpoint kinase 2 (CHK2), cell division control protein 2 (CDC2), and cyclin B1 was investigated using qRT-PCR and Western blot analysis. Results indicated the inhibition of cancer cell growth following exposure to AEAB. In addition, AEAB induced the accumulation of G2/M-phase cells in cancer cell through the disassociation of CDC2/cyclin B1 complex. Our findings suggested that, in addition to the known effects of AEAB in NPC prevention, it may have antitumor activities against NPC cells. In conclusion, AEAB inhibits the growth of and induces mitotic delay in cancer cells, supporting its use as an anticancer agent.

Phytochemical Screening, Alpha-Glucosidase Inhibition, Antibacterial and Antioxidant Potential of Ajuga bracteosa Extracts.

BACKGROUND: Ajuga bracteosa, a medicinal herb, is used by local community to cure a number of diseases such as inflammation, jaundice bronchial asthma, cancer and diabetes. OBJECTIVES: The aim of present work was to evaluate the antioxidant potential, in vitro antidiabetic and antimicrobial effects of A. bracteosa. METHODS: n-hexane, ethyl acetate, chloroform, acetone, methanol and aqueous extracts of Ajuga bracteosa roots, were prepared via maceration. Antibacterial activity was carried out by agar well diffusion method. Quantitative and qualitative phytochemical screening was done. The antioxidant activity was determined by iron (II) chelating activity, iron reducing power, DPPH, and ABTS free radical scavenging methods, Antidiabetic activity was evaluated through inhibition of α-glucosidase assay. RESULTS: Phytochemical analysis showed the presence of phenols, flavonoids, tannins, saponins, quinines, terpenoids, xanthoproteins, glycosides, carbohydrates, steroids, phytosterols and amino acids. DPPH and ABTS potential values were recorded as 61.92% to 88.84% and 0.11% to 38.82%, respectively. Total phenolic and total flavonoid contents were expressed as gallic acid and rutin equivalents. Total iron content was expressed as FeSO4 equivalents. Chloroform and n-hexane extracts showed significant enzyme inhibition potential with IC50 values of 29.92 µg/ml and 131.7 µg/ml respectively. Aqueous extract showed maximum inhibition of E. coli, S. typhimurium, E. amnigenus, S. pyogenes, and S. aureus, (18.0±1.0 mm, 12.5±0.7 mm, 17.0±0.0 mm, 11.0±0.0 mm and 15.3±2.0 mm mm), respectively. Similarly, n-hexane extract showed maximum inhibition of E. coli, E. amnigenus, S. aureus (11.6±1.5 mm; 11.3±1.5 mm; 13.3±0.5 mm). This study also shows that n-hexane, chloroform, ethyl acetate and aqueous extracts of A. bracteosa root possess α-glucosidase inhibitory activities and therefore it may be used as hypoglycemic agents in the management of postprandial hyperglycemia. CONCLUSION: Ajuga bracteosa root extracts may provide a basis for development of antioxidant, antimicrobial and antidiabetic drugs.

Antibacterial activity of 14, 15-dihydroajugapitin and 8-o-acetylharpagide isolated from Ajuga bracteosa Wall ex. Benth against human pathogenic bacteria.

Ajuga bracteosa Wall ex. Benth. (Lamiaceae) commonly known as Bungle Weed has been in use since ancient times and is mentioned Ayurvedic literature. The upper ground parts of the plant are used for treatment of various diseases. The weed is credited with astringent, febrifugal, stimulant, aperient, tonic, diuretic and depurative properties and is used for the treatment of gout and rheumatism, palsy and amenorrhoea. Two compounds 1) 14, 15-dihydroajugapitin and 2) 8-o-acetylharpagide were isolated from the aerial parts of the plant and tested for antibacterial activity against various pathogenic bacteria by agar well diffusion method. Compound 1 and 2 showed maximum antibacterial activity against Escherichia coli with zone of inhibitions of 25. 0 ± 1.4 mm and 22.6 ± 0.9 mm respectively. The MIC value of compound 1 and 2 ranged between 500 and 1000 µg/ml. It could be concluded that both compounds isolated from the aerial parts of Ajuga bracteosa possess antibacterial activity against pathogens.

Evaluation of Ajuga bracteosa for antioxidant, anti-inflammatory, analgesic, antidepressant and anticoagulant activities.

BACKGROUND: Ajuga bracteosa has been extensively used traditionally for the treatment of a variety of diseases. The aim of the study was to scientifically validate the wide-scale exploitation of A. bracteosa in folk medicine various in vitro and in vivo assays. Moreover, these activities were related to the intrinsic biologically active phytoecdysteroids of A. bracteosa. METHODS: Aerial and root parts of A. bracteosa were first extracted separately with chloroform (AbCA and AbCR) and the residue was again extracted with methanol (AbMA and AbMR). Total flavonoid and phenolic contents were assayed as quercetin (QE) and gallic acid equivalents (GAE), respectively. These extracts were analyzed for in vitro antioxidant assessment including DPPH and H2O2 (% inhibition of free radicals), and reducing power and phosphomolybdenum methods (ascorbic acid equivalents AAE mg/g DW). Further, these extracts were assayed in vivo in separate groups of Sprague-Dawley rats for carrageenan induced rat paw edema inhibition, hotplate antinociception, forced swim antidepression and anticoagulation. Dose of each crude extract and standard drug given to rats was 200 mg/Kg- and 10 mg/10 mL/Kg body weight respectively. Plant extracts and standard drugs were administered orally, 60 min prior to the conduction of assays. Moreover, biologically active phytoecdysteroids were screened in A. bracteosa with the help of RP-HPLC. RESULTS: AbMA represented highest values of flavonoids (QE 1.98 % DW) and phenolic contents (GAE 5.94 % DW), significantly scavenged DPPH radicles (IC50 36.9) and reduced ferric ions with 718.4 mg ascorbic acid equivalent/g (AAE). Highest total antioxidant capacity was expressed by AbMR (927 mg AAE) with an IC50 value 19.1 µg/mL. The extracts which were found potent anti-oxidants, were also good at in vivo activities. AbMA significantly reduced edema in all the three hours of treatment (67.9, 70.3 and 74.3 %). AbMA also showed maximum nociceptor suppression in analgesic assay by delaying the time to start licking of paws in rats (57.7 ± 4.9 s). In addition, maximum anti-coagulation was also exhibited by AbMA (89.3 s), while all extracts were found strong antidepressants (≤15.66 s immobility time). Screening of biologically active phytoecdysteroids revealed the presence of 20-hydroxyecdysone (20-HE), makisterone (MKA), cyasterone (CYP) and ajujalactone (AJL). Total phytoecdysteroid content found in A. bracteosa was 1232.5 µg/g DW and 20-HE was most abundant (1232.5 µg/g DW) as compared to other phytoecdysteroids. CONCLUSION: Based on the tested in vitro and in vivo activities, AbMA was found to be a promising bioactive extract. These activities can be attributed to the intrinsic polyphenols and phytoecdysteroids contents of A. bracteosa.

Seasonal and geographical impact on the morphology and 20-hydroxyecdysone content in different tissue types of wild Ajuga bracteosa Wall. ex Benth.

Ajuga bracteosa is an endangered medicinal herb which contains several natural products of therapeutic importance like 20-hydroxyecdysone (20-HE). As geography and habitat play a crucial role in the metabolism and morphology of a plant, the present study was aimed at evaluating the impact of phytogeography, season and tissue type on morphology and 20-HE content of A. bracteosa. The results revealed large morphological variations in various ecotypes of A. bracteosa. However, plants from the same altitude, regardless of their phytogeography, represented similar morphology. Effect of habitat on 20-HE content remained non-significant except for Karot (1608µg/g) and Kahuta (728µg/g). Effect of tissue types was significant (p value <0.016) for 20-HE content and followed ascending order: rootspring (1071µg/g)>summer (617µg/g). The aerial tissue types contained more 20-HE content in all seasons; especially during winter its amount radically rose in flowers (µ=2814µg/g). The aerial portion of Karot ecotype harvested in winter offers a valuable source of 20-HE. To confirm the effect of low temperature on 20-HE content, profiling of A. bracteosa raised in vitro at different temperature regime was carried out. On the basis of these results we hypothesize that chilling cold hampers vegetative growth and triggers stress induced 20-HE accumulation as a defense response.

In-vitro cytotoxicity study of methanolic fraction from Ajuga Bracteosa wall ex. benth on MCF-7 breast adenocarcinoma and hep-2 larynx carcinoma cell lines.

OBJECTIVE: Ajuga bracteosa Wall ex Benth (Labiatae) is popularly known in India as Neelkanthi. A decoction of the leaves, flowers, and barks is used in India for the treatment of cancer including diabetes, malaria, and inflammation etc. The main objective of this study is to investigate the cytotoxic potential of Ajuga bracteosa. MATERIALS AND METHODS: Successive solvent extraction of Ajuga bracteosa in petroleum ether, methanol, and water extracts was done. These extracts were tested against human breast adenocarcinoma (MCF-7) and larynx carcinoma (Hep-2) tumor cell lines, using the thiazolyl blue test (MTT) assay. RESULTS: The methanolic fraction of Ajuga bracteosa had shown the significant results against MCF-7 and Hep-2 tumor cell lines. The methanolic, petroleum ether and aqueous extract from Ajuga bracteosa, presented an IC50 value at 24 h of 10, 65, 70 µg/ml and 5, 30, 15 µg/ml on MCF-7 and Hep-2 cells, respectively. Steroids compounds namely ß-sitosterol and unknown constituents were identified in the most active methanol extract of Ajuga bracteosa wall ex Benth. These known and unknown compounds exhibited cytotoxic potential against MCF-7 and Hep-2 cancer cells. CONCLUSION: Among all the tested extracts, methanolic extract can be considered as potential sources of anti-cancer compounds. Further studies are necessary for more extensive biological evaluations.