Holm Oak (Quercus ilex subsp. ballota (Desf.) Samp.) Bark Aqueous Ammonia Extract for the Control of Invasive Forest Pathogens.

Holm oak (Quercus ilex subsp. ballota (Desf.) Samp.) bark is a commonly used remedy to treat gastrointestinal disorders, throat and skin infections, hemorrhages, and dysentery. It has also been previously reported that its methanol extracts possess antibacterial activity, which can be related to the richness of Quercus spp. extracts in phenolic compounds, such as flavonoids and tannins. However, there is no information on the antifungal (including oomycete) properties of the bark from Q. ilex or its subspecies (ilex and ballota). In this work, we report the characterization of the aqueous ammonia extract of its bark by FTIR and GC-MS and the results of in vitro and ex situ inhibition tests against three phytopathogens. The main phytochemical components identified were inositols (19.5%), trans-squalene (13%), 4-butoxy-1-butanol (11.4%), gulopyranose (9.6%), lyxose (6.5%), 2,4-dimethyl-benzo[H]quinoline (5.1%), catechol (4.5%), and methoxyphenols (4.2%). The efficacy of the extract in controlling forest phytopathogens was tested in vitro against Fusarium circinatum (responsible for pitch canker of Pinus spp.), Cryphonectria parasitica (which causes chestnut blight), and Phytophthora cinnamomi (which causes 'root and crown rot' in a variety of hosts, including Castanea, conifers, Eucalyptus, Fagus, Juglans, Quercus, etc.), obtaining EC90 values of 322, 295, and 75 µg·mL-1, respectively, much lower than those attained for a commercial strobilurin fungicide (azoxystrobin). The extract was further tested ex situ against P. cinnamomi on artificially inoculated, excised stems of 'Garnem' almond rootstock, attaining complete protection at a dose of 782 µg·mL-1. The results suggest that holm oak bark extract may be a promising source of bioactive compounds against invasive forest pathogens, including the oomycete that is causing its decline, the so-called 'seca' in Spain.

Antioxidant, Antimicrobial and In Silico NADPH Oxidase Inhibition of Chemically-Analyzed Essential Oils Derived from Ballota deserti (Noë) Jury.

Ballota deserti (Noë) Jury (B. deserti) is a medicinal plant used in Ayurvedic medicine. The chemical composition, antioxidant, antibacterial, and antifungal properties of essential oils from B. deserti (EOBD) against drug-resistant microorganisms were examined in this work. Hydrodistillation was used to extract EOBD, and gas chromatography-mass spectrometry was used to identify its constituents. Ferric reducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and total antioxidant capacity (TAC) were used to assess the antioxidant effect of EOBD. The disc diffusion agar and the microdilution tests were used in the assessment of the antibacterial properties of EOBD against clinically resistant pathogenic microorganisms. An in silico approach was used to evaluate the inhibitory potential of EOBD against NADPH oxidase. The yield of EOBD was 0.41%, and was primarily composed of linalool (37.82%), cineole (12.04%), and borneol (11.07%). EOBD had good antioxidant potency, with calculated values of 19.82 ± 1.14 µg/mL (DPPH), 64.78 ± 5.21 µg/mL (FRAP), 996.84 ± 20.18 µg EAA/ mg (TAC). Both Gram-negative and Gram-positive bacteria were inhibited by EOBD with inhibition zones ranging from 17.481.75 mm to 28.471.44 mm. EOBD exhibited MICs ranging from 10.78 g/mL to 22.48 g/mL when tested against bacteria using the minimum inhibitory concentration (MIC) assay. Similarly, impressive antifungal activity was observed against fungal strains with inhibition zone widths ranging from 16.761.83 to 36.791.35 mm. Results of MICs assay against fungi showed that EOBD had MICs values ranging from 15.32 ± 1.47 to 23.74 ± 1.54 µg/mL. Docking results showed that thujone, o-cymene, and butanoic acid contained in EOBD possessed strong activity against NADPH oxidase, with glide scores of -5.403, -5.344, and -4.973 Kcal/mol, respectively. In light of these findings, the EOBD may be seen as a potential source of chemical compounds with significant biological capabilities that can be advantageous as natural antioxidants and develop an effective weapon against a wide range of pathogenic bacteria.

Isolation, reactivity, pharmacological activities and total synthesis of hispanolone and structurally related diterpenes from Labiatae plants.

Hispanolone is a furolabdane diterpene isolated from Ballota hispanica, whose natural product chemistry has been summarized and updated here, including several aspects associated with the isolation, structure determination, hemisynthesis, total synthesis, and pharmacology, and related hispanolone diterpenoids that have attracted the interest of different laboratories from diverse perspective and expertise in the last forty-two years.

A Review of the Phytochemistry, Traditional Uses, and Biological Activities of the Genus Ballota and Otostegia.

The 2 genera Ballota and Otostegia, belonging to the Lamiaceae family, are closely related taxonomically and found mainly in the Mediterranean area, Middle East, and North Africa. Since ancient times, they have been largely employed in traditional medicine for their biological properties such as antimicrobial, anti-inflammatory, antispasmodic, insecticidal, anti-malaria, etc. Phytochemical investigations of Ballota and Otostegia species have revealed that diterpenoids are the main constituents of the genera. A large number of flavonoids and other metabolites were also identified. This review, covering literature from 1911 up to 2018, includes traditional uses, chemical profiles (both of volatile and nonvolatile metabolites), and biological properties of all the taxa of these 2 genera studied to date.

In vitro antimicrobial, antiprotozoal activities and heavy metals toxicity of different parts of Ballota pseudodictamnus (L.) Benth.

The study was done to check the antimicrobial and antiprotozoal activity of different parts of Ballota pseudodictamnus (L.) Benth. These activities were then compared with the heavy metals toxicity of different parts, which plants accumulate in different concentrations in different parts. In in-vitro antileishmanial results ethanolic extract, chloroform and ethyl acetate fractions in roots of Ballota pseudodictamnus (L.) Benth showed antileishmanial activity. The ethanol, n-butanol and ethyl acetate fraction in stem revealed inhibition of amastigote form of leishmania. The ethanolic extract, chloroform, and n-butanol fraction in leaves showed inhibition of leishmanial parasite. In heavy metals study, Chromium was above permissible value in all parts except in leaves. Nickel was above WHO limit in roots. Cadmium and lead were beyond permissible limits in entire plant parts. Results revealed that different parts of the plant have different inhibition properties. So each part of plant should be checked for antimicrobial and antiprotozoal assay separately. It is concluded that various metals accumulates with miscellaneous concentrations in different plant parts.

Chemical Composition of Ballota macedonica Vandas and Ballota nigra L. ssp. foetida (Vis.) Hayek Essential Oils - The Chemotaxonomic Approach.

The essential oils isolated from fresh aerial parts of Ballota macedonica (two populations) and Ballota nigra ssp. foetida were analyzed by GC and GC/MS. Eighty five components were identified in total; 60 components in B. macedonica oil (population from the Former Yugoslav Republic of Macedonia), 34 components in B. macedonica oil (population from the Republic of Serbia), and 33 components in the oil of B. nigra ssp. foetida accounting for 93.9%, 98.4%, and 95.8% of the total oils, respectively. The most abundant components in B. macedonica oils were carotol (13.7 - 52.1%), germacrene D (8.6 - 24.6%), and (E)-caryophyllene (6.5 - 16.5%), while B. nigra ssp. foetida oil was dominated by (E)-phytol (56.9%), germacrene D (10.0%), and (E)-caryophyllene (4.7%). Multivariate statistical analyses (agglomerative hierarchical cluster analysis and principal component analysis) were used to compare and discuss relationships among Ballota species examined so far based on their volatile profiles. The chemical compositions of B. macedonica essential oils are reported for the first time.

ANTIOXIDANT ACTIVITY OF TISSUE CULTURE-RAISED BALLOTA NIGRA L. PLANTS GROWN EX VITRO.

Antioxidant properties and total phenolic and flavonoid contents were evaluated in methanolic extracts of shoots from Ballota nigra plants initiated in vitro (from nodal explants) and in vivo (from seeds). The plants were grown in greenhouse and in the field, and were analyzed at the vegetative and flowering stages. The shoot extract of wild-grown plants of B. nigra was also investigated. The results indicate that antioxidant potential of the B. nigra extracts seems to be due to their scavenging of free radicals (DPPH assay) and metal reducing (FRAP test), while they were less effective at the prevention of linoleic acid peroxidation (LPO test). The extracts from shoots of in vitro derived plants were found to exhibit the greatest antioxidant properties. The extracts were also characterized by the highest content of phenolic compounds and their level was affected by plant developmental stage. The extracts of shoots collected at the flowering period exhibited higher amounts of phenolics and flavonoids than in the extracts of immature plants. A close correlation between the total phenolic content and flavonoid content and antioxidant activity using the DPPH and FRAP assays was obtained. The results of the present study suggest the use in vitro-derived plants of B. nigra instead of using wild plants for pharmaceutical purposes.

Investigation of antitussive and toxicological activity of Ballota limbata in mice.

CONTEXT: Ballota limbata Benth. (Lamiaceae) (syn, Otostegia limbata Hook.f.) is a species grown in the North West Frontier Province and the lower hills of West Punjab, Pakistan. Ballota species are renowned for their antispasmodic, antiulcer, diuretic, vermifuge, and especially sedative effects. However, little is known about the biological activity of B. limbata. OBJECTIVE: Evaluation of antitussive activity and safety profile of dried B. limbata extract. MATERIALS AND METHODS: Whole air-dried plants were partitioned with various solvents and the butanol fraction was subjected to antitussive evaluation using a sulfur dioxide (SO(2))-induced cough model in mice. Codeine and dextromethorphan were used as positive control. Safety profile of the testing material was established using standard toxicity tests. RESULTS: B. limbata extract inhibited cough provoked by SO(2) gas in mice in a dose-dependent manner. The extract exhibited maximum protection against SO(2)-induced cough after 60 min of administration. B. limbata offered maximum cough suppressive effects, that is, number of coughs during 60 min was 11.66 ± 1.2 (mean ± SEM), after s.c. administration of 800 mg/kg, as compared with codeine 10 mg/kg, s.c., dextromethorphan 10 mg/kg, s.c., and saline showing a frequency of cough of 11.75 ± 1.18, 12.25 ± 0.83, and 46.25 ± 1.52, respectively. LD(50) value of B. limbata was greater than 5000 mg/kg. No sign of neural impairment was observed at antitussive doses and the extract has been well-tolerated at higher doses. DISCUSSION AND CONCLUSION: This study demonstrates that the extract of B. limbata has shown strong cough suppressive effect in mice without yielding any notable toxicity.

Micromorphological and phytochemical survey of Ballota acetabulosa (L.) Benth.

Within the Open Science project entitled 'Botanic Garden, factory of molecules', a multidisciplinary study approach was applied to Ballota acetabulosa (L.) Benth., at the Ghirardi Botanic Garden (Toscolano Maderno, BS, Italy). Micromorphological and histochemical investigations were performed on the secreting structures of the vegetative and reproductive organs under light, fuorescence and electronic microscopy. Concurrently the characterization of the volatiles spontaneously emitted from leaves and flowers were examined. Four trichome morphotypes were identified: peltate and short-stalked, medium-stalked and long-stalked capitate trichomes, each with a specific distribution pattern. The histochemical analysis was confirmed using ultrastructural observations, with the peltates and long-stalked capitates as the main sites responsible for terpene production. The head-space characterization revealed that sesquiterpene hydrocarbons dominated both in leaves and flowers, with γ-muurolene, ß-caryophyllene and (E)-nerolidol as the most abundant compounds. Moreover, a comparison with literature data concerning the ecological roles of the main compounds suggested their dominant roles in defence, both at the leaf and flower level. Hence, we correlated the trichome morphotypes with the production of secondary metabolites in an attempt to link these data to their potential ecological roles. Finally, we made the obtained scientific knowledge available to visitors of the Botanic Garden through the realization of new labelling dedicated to B. acetabulosa that highlights the 'invisible', microscopic features of the plant.

Determination of the antimicrobial and antioxidative properties and total phenolics of two "endemic" Lamiaceae species from Turkey: Ballota rotundifolia L. and Teucrium chamaedrys C. Koch.

This study was designed to examine the in vitro antimicrobial and antioxidant activities and the amount of total phenolics of the methanol extracts of Ballota rotundifolia L. and Teucrium chamaedrys C. Koch. In the case of antimicrobial activity tests, polar sub-fractions of the methanol extracts of plant species exhibited weakest antimicrobial activity when compared with the non-polar ones. While, non-polar sub-fraction of B. rotundifolia showed moderate activity against A. lwoffii, C. perfringens and the yeasts, T. chamaedrys performed excellent activity pattern against all of the tested microorganisms. The sub-fractions were also screened for their possible antioxidant activities by two complementary tests, namely DPPH free radical-scavenging and beta-carotene/linoleic acid assays. Non-polar extracts of the plant species remained inactive in both test systems. On the other hand polar extracts showed remarkable antioxidant activities. In DPPH system, free radical scavenging effect of T. chamaedrys was measured as 18.00 +/- 1.42 microg.mg(-1). It is extremely important to point out that, polar sub-fraction of T. chamaedrys is found as effective as the positive control BHT. Non-polar sub-fraction of T. chamaedrys found to have the highest total phenolic amount (97.12 +/- 1.28 microg/mg). Results obtained from this experiment confirm the relationship between the amount of phenolics and biological activities.

Effects of Ballota nigra on blood biochemical parameters and insulin in albino rats.

Ingestion of aqueous 70% ethanol extract of Ballota nigra (400 mg/kg body weight for 7 days) by albino rats (n=10) was investigated to study its effects on glucose, total cholesterol, triglycerides, aspartate aminotransferase (AST), alanine aminotransferase (ALT), troponin I (TnI), serum creatine kinase (CK), total protein, total bilirubin and blood urea. Ballota nigra extract caused a significant decrease in blood glucose, total serum cholesterol and CK levels. Blood levels of TnI, AST, ALT, triglycerides, total bilirubin, total protein and blood urea were unchanged. The hypoglycemic effect of Ballota nigra extract on albino rats was further investigated by conducting a glucose tolerance test intraperitoneally (IPGTT). Healthy rats that were fasting for 18 hours followed by administration of a dose of 400 mg/kg body weight of the crude extract of Ballota nigra, orally. A significant decrease in blood glucose levels (after 15, 30, and 45 minutes) with a significant increase in serum insulin level (after 15 and 30 minute) was noted. These results suggest that, the crude extract of Ballota nigra have hypoglycemic, insulin-releasing and cholesterol lowering effects in rats.

Effects of Ballota nigra on glucose and insulin in alloxan-diabetic albino rats.

The hypoglycemic effect of Ballota nigra extract on albino rats was investigated. Alloxan-induced diabetes mellitus was accompanied by several fold increases in plasma glucose. Administration of aqueous extract of B. nigra extract significantly reduced glucose in both healthy and diabetic rats. These results suggest that B. nigra possess hypoglycemic effects in rats and therefore, can be useful for the treatment of diabetes mellitus.

Phytochemical profile of three Ballota species essential oils and evaluation of the effects on human cancer cells.

Three Ballota species, Ballota undulata, Ballota saxatilis and Ballota nigra ssp. foetida, were investigated for their cytotoxicity against two human cancer cells, hepatoma HepG2 cell line and breast cancer MCF-7 cell line, and for their antioxidant activity. The chemical composition of essential oils was studied by GC and GC-MS. Sesquiterpenes were the main constituents. The most antiproliferative essential oil against HepG2 cells was B. undulata with a percentage of inhibition of 81.36 ± 3.54 at a concentration of 100 µg/mL, while against MCF-7 cells essential oil from B. saxatilis was the most active with a percentage of inhibition of 24.18 ± 1.13 at a concentration of 100 µg/mL. The antioxidant activity was investigated by DPPH test for all the oils. B. undulata showed the highest antiradical effect, with IC50 value of 529.7 ± 37.4 µg/mL.

Management of insomnia: a place for traditional herbal remedies.

(1) Insomnia should be treated first with non drug measures; this has traditionally involved the use of herbal remedies. (2) About 20 plants are approved in France in the production of medications 'traditionally used' for minor sleep disturbances. Virtually nothing is known of their efficacy or potential dangers. (3) Most of these plants are suspected of toxicity and should therefore be avoided, especially in view of their unproven efficacy. (4) Littleleaf linden, vervain, melissa and orange flower have no demonstrated efficacy but are safe and can therefore be used. Similarly, there are no scientific grounds for rejecting preparations based on hawthorn or passiflora. (5) Available data suggest that valerian extracts have a modest impact on subjective sleep quality; they are nevertheless more effective than a placebo. Valerian products that do not contain valepotriates have no apparent adverse effects. It is best to avoid high-titre alcoholic extracts and powdered valerian root, and to select aqueous extracts and low-titre hydro-alcoholic preparations.

The absolute stereochemistry of a diterpene from Ballota aucheri.

The semi-synthetic transformation of hispanolone, isolated from Ballota africana, into 6beta-hydroxy-15,16-epoxylabda-8,13(16),14-trien-7-one has established an ent-labdane absolute stereochemistry for a diterpene metabolite originally isolated from B. aucheri.

Antioxidant properties of Ballota species growing in Turkey.

In the present study, the antioxidant properties of ethanol extracts of 16 Ballota species belonging to the Lamiaceae family and growing in Turkey on superoxide anion formation and lipid peroxidation were investigated. All extracts inhibited superoxide anion formation but to different extents. The extracts of Ballota antalyense, Ballota macrodonta, Ballota glandulosissima, Ballota larendana, Ballota pseudodictamnus, Ballota nigra subsp. anatolica, Ballota saxatilis subsp. saxatilis, Ballota rotundifolia, and Ballota saxatilis subsp. brachyodonta exhibited remarkable anti-superoxide anion formation with IC(50) values from 0.50 to 0.87 mg/ml. The extracts of Ballota antalyense, Ballota macrodonta and Ballota glandulosissima have the lowest IC(50) values (0.50, 0.51 and 0.51 mg/ml, respectively) which were rather close to the well-known superoxide anion scavenger alpha-tocopherol (IC(50): 0.22 mg/ml). The extracts of Ballota inaequidens, Ballota glandulosissima, Ballota saxatilis subsp. saxatilis, Ballota macrodonta and Ballota antalyense inhibited lipid peroxidation with IC(50) values from 12 to 20 mg/ml. The extracts of Ballota inaequidens (IC(50): 12 mg/ml) and Ballota glandulosissima (IC(50): 15 mg/ml) exhibited the strongest inhibitory effects on lipid peroxidation compared with alpha-tocopherol (IC(50): 3 mg/ml). The results show that Ballota glandulosissima is the best antioxidant source among these 16 Ballota species.

Hepatoprotective and anti-inflammatory activities of Ballota glandulosissima.

Water extract of Ballota glandulosissima Hub.-Mor & Patzak (Lamiaceae) (BG) was investigated for anti-inflammatory activity using the carrageenan-induced rat paw oedema test and for hepatoprotective effect on carbon tetrachloride (CCl(4))-induced hepatotoxicity in rats. Biochemical parameters of hepatic damage such as serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and bilirubin concentrations were determined. CCl(4) (0.8 mL/kg i.p. for 7 days) treatment increased the serum AST, ALT, ALP and bilirubin levels significantly as compared to controls. Treatment of animals with BG (100 mg/kg, i.p.) +CCl(4) (0.8 mL/kg i.p.) for 7 days significantly ameliorated the levels of AST, ALT and ALP elevated by the CCl(4) treatment alone. The results of biochemical tests were also confirmed by histopathological examination. BG together with CCl(4) treatment decreased the balloning degeneration but did not produced apoptosis of hepatocytes, centrilobular and bridging necrosis observed in the CCl(4) treatment alone. BG, at 100 mg/kg per os, showed a significant reduction (34.22%) in rat paw oedema induced by carrageenan. The reference anti-inflammatory drugs etodolac (50 mg/kg, p.o.) and indomethacin (3 mg/kg, i.p.) significantly reduced the oedeme by 43.42 and 95.70%, respectively. The present study reveals that the water extract of Ballota glandulosissima possesses promising protective activity against CCl(4) induced hepatic damage and anti-inflammatory activity in rats.

Limbetazulone: a new 'decahydro-8-oxanaphtho[2,1-f]azulen-7-one' diterpenoid from Ballota limbeta, and occurrence of two conformational isomers in the crystal.

A new diterpenoid, limbetazulone (= (3S,4S,4aR,12bS)-1,2,3,4,4a,5,6,11,12,12b-decahydro-3-hydroxy-4-(hydroxymethyl)naphtho[1',2': 5,6]cyclohepta[1,2-b]furan-7(7H)-one; 1), with the very rare 'naphtho[2,1-f]azulene-7-one' skeleton, was isolated from the aerial parts of the Asian medicinal plant Ballota limbeta. Its structure was established by extensive spectroscopic investigations, especially 1D and 2D NMR. X-Ray diffraction studies showed the presence of two conformational isomers (1a and 1b) in the crystal.

In vitro antimicrobial and antiprotozoal activities, phytochemical screening and heavy metals toxicity of different parts of Ballota nigra.

The study was done to assess the phytochemicals (flavonoids, terpenoids, saponins, tannin, alkaloids, and phenol) in different parts (root, stem, and leaves) of Ballota nigra and correlated it to inhibition of microbes (bacteria and fungi), protozoan (Leishmania), and heavy metals toxicity evaluation. In root and stem flavonoids, terpenes and phenols were present in ethanol, chloroform, and ethyl acetate soluble fraction; these were found to be the most active inhibiting fractions against all the tested strains of bacteria, fungi, and leishmania. While in leaves flavonoids, terpenes, and phenols were present in ethanol, chloroform, and n-butanol fractions which were the most active fractions against both types of microbes and protozoan (leishmania) in in vitro study. Ethanol and chloroform fractions show maximum inhibition against Escherichia coli (17 mm). The phytochemical and biological screenings were correlated with the presence of heavy metals in selected plant Ballota nigra. Cr was found above permissible value (above 1.5 mg/kg) in all parts of the plant. Ni was above WHO limit in B. nigra root and leaves (3.35 ± 1.20 mg/kg and 5.09 ± 0.47 mg/kg, respectively). Fe was above permissible value in all parts of B. nigra (above 20 mg/kg). Cd was above permissible value in all parts of the plant (above 0.3 mg/kg). Pb was above WHO limit (above 2 mg/kg) in all parts of Ballota nigra.

Essential oil composition and antifungal activity of aerial parts of Ballota nigra ssp foetida collected at flowering and fruiting times.

The present study reports the results of gas chromatographic-mass spectrometric (GC/MS) analyses of the essential oils from the aerial parts of Ballota nigra L. ssp foetida (Lamiaceae) collected at flowering and fruiting times, as well as their in vitro antifungal activity against nine plant pathogenic fungi. Moreover, the essential oils were evaluated for their antifungal activity using the agar dilution method, and also MICs (minimum inhibitory concentrations) and MFCs (minimum fungicidal concentrations) were determined. The major compounds identified in the flowering and fruiting aerial parts oils respectively were beta-caryophyllene (22.6% and 21.8%), caryophyllene oxide (18.0% and 20.5%) and germacrene-D (16.5 and 13.1%). The oils showed in vitro antifungal activity against some species of Fusarium, Botrytis cinerea, and Alternaria solani. Our study indicates that the oil of B. nigra ssp foetida could be used as a control agent for plant pathogenic fungi in natural formulations.