Reviewer’s Comments TITLE: CHEMICAL COMPOSITION AND ANTIMICROBIAL ACTIVITY OF THE ESSENTIAL OILS OF FOUR VARIETIES OF LIPPIA MULTIFLORA IN BENIN

Through this work, we have studied the chemical composition of the essential oils extracted from the leaves by gas chromatography and gas chromatography coupled with mass spectrometry of Lippia multiflora harvested in the regions of Kétou, Savalou, Bohicon and Mono and tested by the well diffusion method against pathogenic microorganisms. The essential oils studied are terpene compounds, aromatic compounds, aliphatic compounds and other natural substances. The inhibition zone diameters determined allowed us to evaluate their degree of germ sensitivity of the strains tested. Essential oils extracted from Lippia multiflora harvested in these areas have the most pronounced antimicrobial activity. In total, the essential oils tested have different and specifically a degree of sensitivity against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Proteus mirabilis A24974, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa except that harvested in the Savalou who does not have no degree of sensitivity on Pseudomonas aeruginosa. This work paves the way for food preservation with extracted natural substances and also the formulation of natural antimicrobials for this fact.


INTRODUCTION
For thousands of years, plants have been occupied a prominent place in the life of man. All known civilizations have used either wild or cultivated plants to provide for their basic needs: food, shelter, clothing and also for their medical needs. The use of medicinal plants has been evolved gradually with the changing needs of humans. Over the centuries, a first distinction has been made between edible and toxic plants. Subsequently, accumulated empirical knowledge has been enabled humans to take plants as the essential source of drugs. The study of this knowledge by modern science gradually reveals some secrets of nature that allow man to continue his evolution. Until the beginning of the 20th century, almost all medicines were herbal. Even today, modern medicine is highly dependent on plants whose therapeutic virtues have been confirmed. Whatever parts and shapes plants use, they are extremely rich in complex chemical structures. The metabolism of plants contains thousands of different constituents, which belong to very different chemical families, such as alkaloids, phenols, flavonoids, terpenoids, steroids. These secondary metabolites, extraordinarily diversified, are widely exploited in various fields: in the culinary field as dyes and flavors, in the medicinal field as antibiotics, antioxidants, drugs, etc., and in the agricultural field as pesticides. These are secondary metabolites of aromatic plants containing in high proportion very active molecules with antibacterial, insecticidal, fungicidal, acaricidal and cytotoxic properties 10 .
Food-borne diseases are nowadays public health problems 23 notwithstanding all the modern innovations in the direction of the improvement and the performance of the techniques and the hygiene of the production of the food. It has been estimated that more than 30% of the populations in industrialized countries suffer from these diseases every year, and more particularly in the year 2000, two million deaths from diarrheal diseases worldwide 9 .
Microbial activity was the primary mode of food deterioration and many microorganisms involved in these food-borne conditions most often contribute to the loss of quality and safety.
In fact, essential oils (HE) are the most exploited thanks to their broad spectrum of known biological activities. Many volatile compounds of essential oils are nowadays common ingredients of pharmaceutical preparations such as pule gone, menthol and thymol 18 ; αpinene, a highly demanded ingredient in the international market, was used in the manufacture of vitamin E 12 . Numerous studies have made it possible, on the one hand, to systematically study essential oils extracted from aromatic plants commonly used in traditional pharmacopoeia in Benin 5 and 4 . 3 , and on the other hand to high light the effectiveness of certain essential oils in the context of integrated protection against certain insect pests of cereal stocks 16 . However, prior to our work, and to our knowledge, no scientific study in Benin has focused on testing the efficacy of volatile extracts of aromatic plants against pathogenic microorganisms and alterations identified in foods. Faced with the resistance of microorganisms to synthetic antibiotics, we undertook this study to propose a cheaper and more credible alternative solution with aromatic plants used in traditional medicine. Our choice, following an ethnobotanical survey, focused on Lippia multiflora 11 . The present study therefore aims to analyze the chemical compositions of the volatile extracts of the retained aromatic plants and to test their effectiveness against certain pathogenic and alteration microorganisms identified in staple foods in Benin. It was a question, specifically to determine the antibacterial activities of the extracts from the diameters of zones of inhibition around the wells vis-à-vis microorganisms tested. The search for these compounds was launched with the aim of discovering new effective therapies against diseases that have not been treated, and to reduce the use of synthetic products that are harmful to humans and their environment. Like antibiotics, antioxidants and pesticides. Despite attempts to list plants and their extracts, the number of species studied remains relatively low given the growing need for natural compounds of aromatic and / or medicinal interest.

Plant material
It consists of plant material (Lippia multiflora), microbial strains (bacteria, yeast and mold),

Biological and chemical diagnostic equipment:
The equipment used in the laboratory consists of conventional glassware sterile single-use plates of 90mn diameter, a micropipette P100 Gilson brand and culture media. The culture media and reagents used in the microbiological diagnosis come from BIORAD, MERCK, OXOID, BASINGSTORE, BIOMERIEUX and DifCO laboratories. A 24-hour pure colony portion from the Mueller Hinton medium of each strain was emulsified in 5ml of physiological saline to obtain a turbidity of 0.5 on the Mc Farland scale. The chemical diagnostic equipment is a GC, a spectrometer, Clevenger for extraction of essential oils.

METHODS
The survey was carried out using the method of 21

Extraction of oils and calculation of yields
About 200 g of plant material from each plant were each subjected to hydrodistillation with a Clevenger type apparatus for 3 hours 3 , 14

Chemical composition of essential oils Analysis by gas chromatography
The oils were analyzed using a VARIAN CP.3380 gas chromatograph equipped with two capillary columns (apolar and polar) and connected to a Varian integrator (Model C-R4A°); 22 .

Technical characteristics of the equipment
Identification from retention indices and mass spectra. All essential oils were analyzed by

Evaluation of the antibacterial activity of essential oils Isolation and purification of microbial strains:
The clinical strains were isolated from stool samples at the National Public Health Laboratory of Benin.

Sensitivity test by well diffusion:
Each inoculum was seeded by swabbing onto petri dishes containing Mueller Hinton agar.
Using the sterile pasteur pipette tip 6 mm diameter wells were dug. Then using a cone and a micropipette 50µl of each extract were deposited in the wells previously dug. A well containing sterile distilled water to serve as a negative control. The Petri dishes were left for 1 hour at room temperature for pre-diffusion of the substances, before being incubated at 37°C.
in an oven for 18 hours. After the incubation period, the dishes were examined for measurements of the inhibition zones. The antibacterial activity of the extracts was determined from the diameters of inhibition zones around the wells as shown (Figure 1).

Chemical composition of essential oils:
We will study the chemical compositions of essential oils according to its large botanical family of different regions 14 .
Verbenaceae: This family includes Lippia multiflora (from Kétou, Savalou, Bohicon and Mono).  conditions that change from one region to another, and at the time of harvest 19 . As for the total number of compounds found in Lippia multiflora Kétou was five compounds less than the other Savalou, Djidja and Mono which contain respectively nineteen, fourteen and ten compounds. The essential oil of Lippia multiflora is generally composed of molecules that the pharmaceutical industry often uses. Indeed, the presence of α-pinene compounds, the most common molecule-marking terpene in nature, was an effective molecule for the treatment of respiratory conditions such as colds, coughs or bronchitis. Moreover, thanks to the similarity of its smell with that of fir, it was commonly used in deodorants and cleaning products. On the other hand, the presence of the compound (Z) -β-ocimene was known for its properties and its stabilities notably preventing its oxidation. This composition was different from that previously reported by 1 , 7 , and 13 .
Antimicrobial activities of the investigated essential oils:

CONCLUSION
The chemical composition of the essential oil extracted from the leaves of Lippia multiflora  (Table 3) act differentially on the isolated microbial strains and in the present study, it appears that the Gram + are more sensitive to their effect than the Gram. Through these works, the essential oil of Lippia multiflora offers hope. Indeed, this oil, commonly called "Gambia tea" is an aromatic plant very little known in Benin and used in the treatment of several pathologies in traditional medicine. Bibliographic research has also shown that this plant has several therapeutic virtues. The results of our work confirm these effects and show that this plant has excellent antimicrobial properties.