Optimization of the Extraction of Total Flavonoids and Antioxidant Activity from Lippia multiflora Moldenke (Verbenaceae) Leaves using Experimental Design

The leaves of Lippia multiflora contain secondary metabolites including flavonoids which have an important antioxidant activity. This study aims to optimize the extraction conditions of total flavonoids and the antioxidant activity of these leaves. To achieve this, the Plackett-Burman design was used for the screening of the factors influencing the extraction, then the central composite design was implemented for the optimization itself. The effects of five factors, such as the plant-to-solvent ratio, the nature of the extraction solvent, the extraction time, the extraction method and the size of the L. multiflora leaves, on the extraction of total flavonoids and the antioxidant activity were studied. Results of Plackett-Burman design indicated that factors influencing both flavonoids extraction and antioxidant activity were the solid-liquid ratio and extraction time. The predicted optimal conditions for the highest flavonoids content from L. multiflora leaf with better antioxidant activity were found with aqueous decoction for 30 min with 3.5 g of cut leaves in 100 mL of distilled water. Using the predicted conditions, experimental responses were 87.18 ± 1.03 mg/g QE and 372.34 ± 4.04 µmol/g TE for total flavonoids and antioxidant capacity, respectively. Under the above-mentioned conditions, the experimental results are very close to predicted one. Thus, L. multiflora leaf can be considered as a natural source of flavonoids content with good antioxidant activity.

INFLUENCE OF HARVESTING SITES ON THE ANTIHYPERTENSIF EFFECTS OF ESSENTIAL OILS OF LIPPIA MULTIFLORA MOLDENKE (VERBENACEAE) IN BENIN

A comparative study of the major compounds was carried out despite the great diversity of the constituents characteristic of the essential oil of Lippia multiflora. It was interested to note the imprint that makes each locality with the chemical characters of the gasoline. The variation in the chemical composition of essential oils, was depended on many factors such as the method used, the parts of the plant used, the products and reagents used in the extraction, the environment, the genotype of the plant, the geographic origin, harvest period of the plant, degree of drying, drying conditions, temperature and drying time and the presence of pests, viruses and weeds. The effects of essential oils of Lippia multiflora were evaluated in Wistar rats on arterial pressure (BP) and on heart rate (HR). Administration of L-NAME was caused a signicant increase in SBP in rats from 142.4 mmHg (D0) to 172.4 mmHg (D8) and from 122.8 mmHg (D0) to 138.6 mmHg (D8) respectively for the positive control batch and the batch subsequently treated with essential oil. Administration of the essential oil from D8 to D29 was caused a signicant decrease in the SBPof the rats from 140 mmHg (D8) to 122.4 mmHg (D29). This decrease was markedly lower than that of the untreated rats which is 150.5 mmHg (D29). The administration of L-NAME was caused a signicant increase in the DBPof the rats going from 93.8 mmHg (D0) to 137.5 mmHg (D8) and from 92.5 mmHg (D0) to 136.75 mmHg (D8) respectively for the positive control batch and the batch subsequently treated with essential oil.. The administration of the essential oil from D8 to D29 was caused a signicant decrease in the DBP of the rats going from 136.75 mmHg (D8) to 99.8 mmHg (D29) and nally the administration of L-NAME caused a signicant increase in the MAP of the rats going from 109.25 mmHg (D0) to 159.75 mmHg (D8) and from 99.8 mmHg (D0) to 151.25 mmHg (D8) respectively for the positive control group and the batch then treated with the raw extract). Administration of the essential oil from D8 to D29 was caused a signicant decrease in the AMP of the rats, dropping from 151.25 mmHg (D8) to 114 mmHg (D29) (p <0.05). These effects could be justify the use of this plant in traditional medicine in the treatment of high blood pressure.

Potential of an aqueous extract of Lippia multiflora Moldenke (Verbenaceae) in the treatment of anxiety disorders: Possible involvement of serotoninergic transmission

Ethnopharmacological relevance: The leaves and stems of Lippia multiflora Moldenke (Verbenaceae) are used in traditional medicine to treat depression, anxiety and epilepsy. Aim of the study: The present study investigated the anxiolytic-like effects of Lippia multiflora aqueous extracts in experimental models of anxiety. Materials and methods: Elevated plus maze and open field tests were used to evaluate anxiety and exploratory behaviour. The possible mechanism of action of Lippia multiflora were investigated after pretreatment of animal with agonist and antagonist of serotonin receptors, such as: mCPP, a partial agonist at the 5-HT2C receptors; Way-100635, a selective 5-HT1A receptors antagonist; and DOI, the 5-HT2A/2C receptors agonist. The levels of monoamine neurotransmitters were evaluated in the brain. Results: In the elevated plus maze, Lippia multiflora increased the number of entries into and percentage of time in open arms, and reduced rearing, head dipping, and percentage of time in closed arms. These effects were blocked with the agonist and antagonist of serotonin receptors. In the open field, Lippia multiflora increased crossing and reduced rearing and defecation. The endogenous levels of noradrenaline, dopamine and serotonin in the brain homogenates remain comparable to that distilled water-treated animals. Conclusion: It could be concluded that Lippia multiflora aqueous extracts, used in traditional medicine in Cameroon for the treatment of depression, anxiety and epilepsy seemed to possess anxiolytic properties in mice. These effects might be mediated by serotonin receptors, but not by modulation of the levels of monoamine neurotransmitters when the extract is administered once after one hour.

Biochemical composition and sensory characteristics of infusions of leaves from two morphotypes of Lippia multiflora (verbenaceae) grown in Côte d’Ivoire

Objectives: this study aims to compare composition and sensorial characteristics of infusions of leaves from broad leave morphotype (blmLM) and long leave morphotype (llmLM) of Lippia multiflora (Savannah tea) cultivated in three localities, in order to identify the morphotype and/or locality, which offer the better biochemical composition and organoleptic qualities of tea. Methodology and results: Leaves harvested were dried and pulverized. Infusions were prepared using standard methods and sensorialy analysed by Mossion methods. Elements were determined according to AOAC methods. Phenolics compounds and caffeine was analysed by a standard method. Infusions of blmLM are rich in tannins (12.15-14.26 mg/L), quercetin (0.33-0.95 mg/L) and flavanone (05.75-13.33 mg/L), while those of llmLM are rich in caffeine (19.07-20.14 mg/L) and catechin (08.89-59.56 mg/L). Calcium (50.96- 51.54 mg/L) and magnesium (35.10-37.08 mg/L) contents are highest in infusions of BlmLM, while llmLM infusions are richer in K (97.78-103.46 mg/L) and Na (143.01-156.03 mg/L). Globally, infusions are brown to greenish yellow, astringent, slightly bitter with a pleasant smell. Infusions of Béoumi and Korhogo have a mint scent; those of Béoumi have a scent of lemongrass. Conclusion and Application of results: The qualities of infusions depend on the morphotype and the growing area. Their richness in phenolic compounds, caffeine and essential minerals gives them nutritional, medicinal and pharmacological properties and can be a replacement for ordinary teas sold on the market. This study provides information on the choice of the plant morphotype and the cultivation area according to the needs of the consumer. Consumption of infusions of leaves from Korhogo and Béoumi that are richer in tannins, catechin, quercetin and essential minerals (calcium, magnesium and iron) will be beneficial for human nutrition, bones strengthening and prevention of cardiovascular diseases which represent a public health problem. Taking Lippia multiflora into account in the agricultural and eating habits of farmers will constitute for them a source of income and also a source of permanent raw material for the agro-food processing industries. Keywords: Lippia multiflora, leaves infusion, biochemical composition, sensory characteristics

Effects of Lippia multiflora leaf extract on the semen of Wistar rat

To investigate the effect of Lippia multiflora (Verbanaceae) on the reproductive activity of male Wistar rats, three groups (A,B and C) of rats were treated with 400, 800 and 1600 mg/kg/day of Lippia multiflora leaves extract respectively for 28 days while 5ml/kg of distilled water was given to the control group D. At the end of the experimental period, animals were sacrificed and sperm characteristics, histology of the testes and epididymis were assessed. Lippia multiflora leaf extract caused a significant reduction (P<0.05) in sperm motility, a statistically significant dose dependent reduction in the sperm count and a significant (P<0.05) dose dependent increase in morphological abnormalities of the spermatozoa of the treated rats. Histopathological evaluation of the testis and epididymis revealed varying degrees of degeneration and necrosis of the germinal epithelia cell of the seminiferous tubules, spermiostasis and complete epididymal aspermia. Lippia multiflora leaf extract has significant anti-spermatogenic effects on adult male Wistar rats which could impair reproductive activities in these rats

Chemical Composition and Antibacterial Activity of Lippia multiflora Moldenke Essential Oil from Different Regions of Angola

The purpose of the study was to determine the chemical composition and antibacterial activity of Lippia multiflora Moldenke essential oils (EOs) collected in different regions of Angola. Antibacterial activity was evaluated using the agar wells technique and vapour phase test. Analysis of the oils by GC/MS identified thirty-five components representing 67.5 to 100% of the total oils. Monoterpene hydrocarbons were the most prevalent compounds, followed by oxygenated monoterpenes. The content of the compounds varied according to the samples. The main components were Limonene, Piperitenone, Neral, Citral, Elemol, p-cymene, Transtagetone, and Artemisia ketone. Only one of the eleven samples contained Verbenone as the majority compound. In the vapour phase test, a single oil was the most effective against all the pathogens studied. The principal component analysis (PCA) and hierarchical cluster analysis (HCA) of components of the selected EOs and inhibition zone diameter values of agar wells technique allowed us to identify a variability between the plants from the two provinces, but also intraspecific variability between sub-groups within a population. Each group of essential oils constituted a chemotype responsible for their bacterial inhibition capacity. The results presented here suggest that Angolan Lippia multiflora Moldenke has antibacterial properties and could be a potential source of antimicrobial agents for the pharmaceutical and food industry.

Qualités morpho-physiologiques et évaluation du comportement germinatif des graines du théier des savanes (Lippia multiflora Moldenke)

La domestication de Lippia multiflora, plantes aux multiples vertus en pharmacopée et médecine est un enjeu de taille en Côte d’Ivoire. Cependant, l’insuffisance de semences, due au faible taux de germination des graines, limite l’extension de sa culture. La présente étude avait pour objectif l’extraction, la purification et la caractérisation morpho-physiologique des graines de Lippia multiflora Moldenke (Verbenaceae). Il s’agissait plus spécifiquement de déterminer la pureté spécifique, le nombre de graines par unité de masse, le diamètre moyen et le taux d’humidité ; le taux de germination des graines de L. multiflora. Aussi, il s’est agi d’étudier l’impact du milieu sur le pouvoir germinatif des graines et de faire un suivi post-germination des plants en milieu réel. Après extraction des graines, des mesures physiques et des tests de germination ont permis de déterminer les caractéristiques morpho-physiologiques de celles-ci. L’étude a montré que le lot de graine étudié a une pureté spécifique de 70%, les graines ont un diamètre moyen de 0,34± 0.1 mm, une teneur en eau de 14±5.4 % et un taux de germination de 42,25%. Les tests de germination après un séjour prolongé dans divers milieux, révèlent qu’un milieu réfrigéré (7° C) confère une plus longue viabilité aux graines. Le suivi post-germination au champ montre une évolution régulière de la hauteur des plants, passant en moyenne de 2,58 cm à 8,8 cm au bout de 3 mois. Sur la même période, le nombre moyen de feuille varie de 4,03 à 21.Mots clés : Lippia multiflora, graine, caractérisation, germination, suivi post-germination. English Title: Morpho-physiological qualities and evaluation of the germination behavior of seeds of the savannah tea tree (Lippia multiflora Moldenke)The domestication of Lippia multiflora, plants with multiple virtues in pharmacopoeia and medicine is a major challenge in Côte d'Ivoire. However, the lack of seed, due to the low germination rate, limits the extension of its cultivation. The purpose of this study was the extraction, purification and morpho-physiological characterization of the seeds of Lippia multiflora Moldenke (Verbenaceae). More specifically, it involved determining the specific purity, the number of seeds per unit mass, the average diameter and the humidity rate; the germination rate of L. multiflora seeds. Also, it was a question of studying the impact of the environment on the germination power of seeds and of making a post-germination follow-up of the plants in real environment. After the seeds extraction, physical measurements and germination tests were carried out to determine the morpho-physiological characteristics. Results showed that the seed lot studied has a specific purity of 70%, an average diameter of 0.34 0.1 mm, a water content of 14 5.4% and a germination rate of 42.25%. Germination tests after a prolonged stay in various environments reveal that a refrigerated area (7 °C) confers a longer viability to the seeds. Post-germination monitoring in the field shows a steady increase in plant height from an average of 2.58 cm to 8.8 cm after 3 months. Over the same period, the average number of sheets varies from 4.03 to 21.Keywords: Lippia multiflora, seed, characterization, germination, post-germination monitoring.

Assessment of the Exposure Risk to Mycotoxins from Stored Maize (Zea mays L.) in Triple Bags with Aromatic Plants Leaves (Lippia multiflora and Hyptis suaveolens) in Côte d’Ivoire

In Côte d’Ivoire, maize is second cereal most cultivated and consumed after rice. In bad storage conditions, corn may be contaminated by mycotoxins (aflatoxin B1, total aflatoxins, ochratoxin A, fuminosin B1, zearalenone). The aim of this study was to assess the exposure risk of these mycotoxins for maize stored in triple bags in presence of aromatic plants leaves (Lippia multiflora and Hyptis suaveolens). The contents of water activity, aflatoxin B1 (AFB1), total aflatoxins (AFT), ochratoxin A (OTA), fuminosin B1 (FB1) and zearalenone (ZEA) were studied and monitored. The levels of AFB1, AFT, OTA, FB1 and ZEA resulted from maize grains treated with plants leaves were significantly lower than those recorded with untreated maize of control bags. The estimated daily intakes in AFB1, AFT, OTA, FB1 and ZEA, deriving with consumption of maize from experimental batches stored for 18 months are respectively 1.69 ± 0.00 - 2.09 ± 0.01 ng/kg bw/day, 8.66 – 10.91 ng/kg bw/day, 1.86 ± 0.01 - 2.47 ± 0.01 ng/kg bw/day, 2.01 ± 0.05 - 3.01± 0.05 ng/kg bw/day and 1.89 ± 0.12 - 3.56 ± 0.04 ng/kg bw/day. These levels are lower than the estimated intakes from maximal reference value (EDILM) for OTA, FB1 and ZEA. For aflatoxin B1 and total aflatoxins, the estimated daily intakes are lower than the estimated intakes from maximal reference value (EDILM) during, respectively, 15 storage months and 10 storage months. However, after 18 storage months, exposure risk of aflatoxin B1 is higher than the estimated intakes from maximal reference value. This inexpensive and easy-to-use treatment should be popularized among farmers

Adsorption of Lippia multiflora essential oil on two surfactant-modified clays: qualitative approach

This paper deals with the adsorption of an essential oil (EO) on a kaolinite-rich clay and a smectite-rich clay. The two clays were modified with a quaternary alkyl ammonium surfactant to create a lipophilic environment for better adsorption of the EO. The preparation of the clay/EO hybrids avoided the use of a slurry and organic solvent. The selected EO was that of Lippia multiflora. This EO has insecticidal properties. The surfactant was trioctyl methylammonium (TOMA). The modified clays were characterized by X-ray diffraction (XRD) and infrared (IR) spectroscopy. The smectite-rich clay displayed greater adsorption of the L. multiflora EO compared to the kaolinite-rich clay. The interlayer space of the kaolinite-rich clay was not affected by the adsorption of the TOMA and/or EO molecules, which suggests that the adsorption in this clay took place on the external surface. By contrast, a significant increase in the interlayer space of the smectite-rich clay was observed, suggesting that the adsorption process of TOMA and/or EO took place on both the external and internal surfaces. The IR analysis showed that the surfactant loading in the interlayer space of the smectite-rich clay introduces a gauche conformation in the alkyl chains. A formulation mixing this local smectite-rich clay and the L. multiflora EO has potential for the manufacture of new biopesticides.