INTRODUCTION
Preface
Throughout history, humans have utilized plants to combat infectious diseases, with scientific research confirming their therapeutic benefits Today, various countries rely on plants to address ailments, including respiratory, gastrointestinal, urinary, and biliary infections Despite advancements in microbiological research and disease control, the rise of drug-resistant bacteria and new pathogenic strains highlights the urgent need for new antibiotics Consequently, the exploration of medicinal plants through advanced technologies is being revisited as a promising strategy to discover novel bioactive agents that can tackle significant public health challenges.
The search for natural active ingredients with high biological activity for medicinal purposes is a growing trend among scientists Various medicinal materials have been utilized to extract valuable compounds, including berberine from Coscinium fenestratum, morphine from Papaver somniferum, and curcumin from Curcuma longa Other notable sources include beta-carotene and lycopene from Mormodica cochichinensis, papain from Carica papaya, and diosgenin from Dioscorea deltoidea Essential active ingredients like quinine, morphine, and strychnine are derived from medicinal herbs and cannot be synthesized chemically.
The evaluation of medicinal herbs requires a thorough analysis of their antibacterial activity, polyphenol content, and antioxidant properties This investigation is crucial for understanding the potential benefits and effectiveness of these herbs in medicinal applications.
Medicinal herbs like lemongrass, cinnamon, and anise have been utilized in folk medicine for their ability to cure diseases, enhance immunity, and improve overall health These herbs show significant promise in pharmaceutical research and development, serving as cost-effective and high-quality alternatives to antibiotics and synthetic chemicals Consequently, we conducted a study to extract and assess the antibacterial properties, polyphenol content, and antioxidant activity of lemongrass, cinnamon, and anise.
Objective and requirements
- Proven antibacterial effect of medicinal herbs lemongrass, cinnamon, and anise on bacteria Bacillus subtilis ATCC 6633, Geobaccillus stearothermophilus ATCC 7953, Staphylococcus aureus ATCC 25023, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 85922,
Escherichia coli ATCC 35218, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, Salmonella ATCC 13311
- Investigation of polyphenol content and antioxidant activity of extracts and essential oils
- Choose the right solvent to use in the extraction process to get the best results
- Investigation of the antibacterial activity of the main active ingredients in medicinal herbs.
LITERATURE OVERVIEW
Potential for development of herbal medicine
According to the World Health Organization (WHO), about 80% of the population in rural and developing countries still rely on herbal medicines for public health care (Phung Tuan Giang, 2017)
The World Health Organization (WHO) reports that over 20,000 species of higher vascular plants and lower branches are utilized for medicinal purposes, with tropical America contributing more than 1,900 species and tropical Asia around 6,500 species of flowering plants The demand for herbal medicines is steadily increasing, prompting global research into the therapeutic mechanisms and chemical compounds found in these plants Many plants exhibit antibiotic properties due to natural compounds such as phenolics, alkaloids, and flavonoids Significant advancements have been made in understanding these natural compounds, leading to their extraction for medicinal use and the synthesis of artificial substances for disease treatment Notable examples include the isolation of Glucoside barbaloid from Aloe vera, which combats tuberculosis, and berberin extracted from the Hoang Lien tree Additionally, the shallot plant contains beneficial compounds like sulfide and sapoin, while Odorin, isolated by Shen-Chi-Shen, demonstrates antibacterial effects with low toxicity to higher animals Chive seeds and Rauvolfia spp also provide alkaloids and antihypertensive agents, respectively, showcasing the diverse medicinal potential of plants.
Vinblastine and vincristine, derived from the periwinkle plant, exhibit both antihypertensive and anti-cancer properties Additionally, digitalin is extracted from the poplar tree (Digitalis spp.), while strophanthin comes from the horned goat tree (Strophanthus spp.) and is used as a heart stimulant.
Despite the rich potential for developing natural herbal drugs due to the abundance of available herbs, the utilization of medicinal plants for community use remains lower than in other countries This highlights the need for increased cultivation and application of these plants (Phung Tuan Giang, 2017).
Overview of medicinal herbs
Figure 2.1 Lemongrass and lemongrass powder
The essential oil content in lemongrass has risen from 0.46% to 0.55% This oil is rich in active ingredients, comprising 65-85% citral and myrcene, which are known for their antibacterial and analgesic properties, along with other components such as citronella, citronellol, and geranilol.
- Lemongrass has the following main effects :
• Detoxify the body: Lemongrass has a diuretic effect and supports the digestive system to work better Therefore, they help the pancreas, kidneys, liver and bladder become cleaner
Lemongrass has been found to be an effective antiseptic, with studies published in a Brazilian medical journal indicating its potential as a treatment for staph infections The essences in lemongrass demonstrate superior antibacterial properties, inhibiting and killing bacteria more effectively than traditional antibiotics.
• Anti-inflammatory: A study published in 2010 said that lemongrass extracts have anti-inflammatory effects, helping to improve symptoms of inflammatory bowel disease
Regular consumption of lemongrass juice may help prevent cancer, as research indicates it can effectively kill cancer cells, including those associated with breast and liver cancer This is attributed to the presence of luteolin in lemongrass essence, an active compound known for its ability to inhibit and slow the growth of cancer cells.
- Scientific name: Cinnamomum cassia (L.) J Presl
Figure 2.2 Cinnamon and cinnamon powder
Cinnamon is renowned for its high antioxidant activity and possesses potent anti-bacterial and anti-fungal properties due to its essential oil Additionally, it is a rich source of fiber, manganese, iron, and calcium, making it a valuable remedy for various health purposes.
• Lowering blood sugar and treating type 2 diabetes
• Enhance memory and cognitive function
Figure 2.3 Anise and anise powder
Anise fruit is rich in essential oil, with a steam extraction process yielding 3-3.5% of a colorless or light yellow liquid essential oil that has a distinctive aroma The primary component of anise essential oil is anethol, which constitutes 80-90% of its composition, along with other compounds such as a-pinene, d-pinene, l-phellandrene, safrol, terpineol, and limonene While anise leaves also contain essential oils, they are present in slightly lower concentrations, and anise seeds are odorless, primarily consisting of fatty oils.
Anethole increases gastric and intestinal motility, soothes abdominal pain, increases respiratory secretion by stimulating secretion cells, can be used as a expectorant
Anise extract alcohol demonstrates significant antibacterial activity against various pathogens, including Staphylococcus aureus, pneumococci, diphtheria bacillus, subtilis bacilli, typhoid, and paratyphoid bacilli Additionally, it effectively inhibits certain fungi responsible for skin diseases.
Overview of bacteria
In this topic, we used 9 bacteria including :
2.3.1 The gram-positive bacteria a Bacillus subtilis
Bacillus subtilis is a small, motile bacillus characterized by its purple Gram-positive staining and size, ranging from 0.5 to 0.8 µm in width and 1.5 to 3 µm in length, often found singly or in short chains This bacterium features 8-12 flagella and produces oval spores, measuring 0.8 to 1.8 µm, which can be located centrally or eccentrically within the cell The spores are formed through germination following spore cracking and exhibit resistance to heat (up to 100°C for 180 minutes), moisture, ultraviolet radiation, pressure, and antiseptics, although they are not acid-resistant Remarkably, these spores can survive for several years to decades.
Culture properties: Bacillus subtilis bacteria grow under aerobic conditions, but can still grow in anoxic environment Optimum temperature is
– Bacillus subtilis bacteria grow on most basic nutrient media:
On Trypticase Soy Agar (TSA) plates, colonies appear round with irregular serrated edges, exhibiting a yellow-gray color and measuring 3-5 mm in diameter After 1-4 days, the surface of these colonies becomes wrinkled and takes on a slightly brown hue.
+ On Trypticase Soy Broth (TSB) broth: bacteria grow, making the medium turbid, forming wrinkled films, sediments, and clumps like clouds at the bottom, hard to dissolve when shaken well
+ On bean sprouts medium - peptone: colonies are round convex, smooth, sometimes spreading, irregular serrated edges, 3-4cm in diameter after 72 hours of culture b Geobacillus stearothermophilus
Geobacillus stearothermophilus, also known as Bacillus stearothermophilus, is a Gram-positive, rod-shaped bacterium belonging to the Bacillus genus This thermophilic bacterium thrives in environments such as soils, hot springs, and ocean sediments, and is known to cause food spoilage It can grow within a temperature range of 30 to 75°C.
Staphylococcus aureus, a member of the Staphylococcus genus, exhibits key characteristics such as being spherical, non-motile, and Gram-positive, with a diameter ranging from 0.5 to 1.5 µm These bacteria are typically arranged in grape-like clusters and possess a cell wall that is resistant to lysozyme but sensitive to lysostaphin, which can disrupt the pentaglycine bridge in staphylococci Additionally, S aureus are facultative anaerobes or aerobes, equipped with the enzyme catalase that decomposes hydrogen peroxide, producing oxygen and water.
Staphylococcus aureus exhibits remarkable adaptability, thriving in a broad temperature range of 7 to 48°C, with optimal growth between 30-45°C, and a pH tolerance of 4.2 to 9.3, favoring 7 to 7.5 This bacterium can survive in environments with over 15% NaCl and demonstrates resilience against high sugar concentrations, although it is inhibited at 60% S aureus shows strong adhesion to various surfaces, enhancing its resistance to drying and filtration Consequently, it is widely distributed, primarily found on human and warm-blooded animal skin, mucous membranes, hair, and nasal passages While S aureus can persist in the air, dust, and water, it is relatively immobile and sensitive to antibiotics and biocides Additionally, it is vulnerable to heat, being killed at 60°C within 2 to 50 minutes, depending on the food type, and is a weak competitor, easily outcompeted by other microorganisms.
S aureus bacteria cause many infectious, purulent and toxic diseases in humans Usually occurs in superficial scratches such as boils, causing many serious infections such as pneumonia, mastitis, phlebitis, meningitis, urinary tract infections and other dangerous diseases such as osteomyelitis, inflammation of the endocardium S aureus is also a major cause of surgical site infections and medical instrument infections S aureus also causes food poisoning because 13 enterotoxins in food
2.3.2 The Gram-negative bacteria a Escherichia coli
E coli (short for Escherichia coli) is a type of bacteria that normally lives in the intestines of humans and animals There are different types of E coli
E coli is a bacterium that naturally resides in our intestines, aiding in the digestion of food However, certain strains of E coli can lead to diarrhea and other health issues when ingested.
Escherichia coli (E coli) is a Gram-negative bacillus, typically measuring 2 to 3 micrometers in length and 0.5 micrometers in width Under adverse conditions, such as exposure to antibiotics, these bacteria can elongate significantly While only a few strains possess a protective shell, the majority are characterized by their hair-like structures and motility.
Culture properties : E coli grows readily on conventional culture media
Some can grow on synthetic media that are very poor in nutrients Aerobic or facultative anaerobic Can grow at temperatures between 5°C and 40°C
E coli exhibits rapid growth under optimal conditions, with a generation time of approximately 20 to 30 minutes Inoculating E coli into a liquid medium results in slight cloudiness after 3 to 4 hours, turbidity after 24 hours, and a thin scum on the surface after two days Over the following days, the bottom of the tube becomes visible On normal agar, colonies can be observed after 8 to 10 hours with a magnifying glass, reaching about 1.5 mm in size after 24 hours The typical colony morphology is S-shaped, although R or M shapes may also be present Additionally, Salmonella enterica is mentioned in the context of bacterial growth.
Species : S enterica Biological characteristics: Salmonella is a bacterium of the
The Enterobacteriaceae family consists of rod-shaped, gram-negative bacilli that are non-spore forming anaerobes These motile bacteria, characterized by their flagella, inhabit the intestinal tract and typically measure between 0.7 µm to 1.5 µm in diameter and 2 µm to 5 µm in length.
Salmonella exhibits both aerobic and facultative anaerobic properties, making it easy to culture It thrives at a temperature of 37°C, with a growth range of 6°C to 42°C and an optimal pH of 7.6, developing well in a pH range of 6 to 9 While it shows good growth in aerobic conditions, its growth is less favorable under anaerobic conditions On normal agar, Salmonella forms round, clear or gray colonies that are smooth and slightly convex in the center, appearing smaller and whiter than E coli colonies, with a diameter of approximately 1 mm.
Salmonella is a bacteria that causes diseases in humans and animals, mainly digestive diseases, causing diarrhea, paratyphoid in pigs, d Pseudomonas aeruginosa
Pseudomonas aeruginosa is a Gram-negative, absolutely aerobic bacterium characterized by its thin, straight or slightly curved shape with rounded ends, measuring 0.5-1.0 x 1.5-3 μm This motile organism features a single flagellum at one end and is non-spore forming, with pili present at the pole, measuring 6 nm in width.
Culture properties: Can grow at 4°C – 41°C, but most suitable is 37°C, prefers neutral pH
Colony morphology: On blood agar, normal agar: Three types of colonies
+ S-shaped colonies: 1-2 mm in diameter, round, smooth, slightly convex after 18-24 hours, beta hemolytic, metallic luster, greenish pigment, special aroma like grape
+ Colony type R: smaller in diameter, rough, convex, often isolated from the outside
+ M-type colonies: mucinous colonies, beta hemolytic
Pseudomonas aeruginosa is a significant pathogen responsible for various organ diseases and hospital-acquired infections This bacterium can survive for extended periods in healthcare settings, including washing water, floors, and on the hands of medical staff It frequently leads to purulent infections in wounds, which can subsequently result in bacteremia as the bacteria enter the bloodstream.
In addition, they can cause urinary tract infections, respiratory infections, and bacterial infections in the graft.
Overview of polyphenol
Polyphenols are a category of compounds naturally found in plant foods, such as fruits, vegetables, herbs, spices, tea, dark chocolate, and wine
Antioxidants play a crucial role in protecting your cells by neutralizing harmful free radicals, which can otherwise lead to serious health issues such as cancer, diabetes, and heart disease (Lien Ai Pham-Huy et al.).
Polyphenols are also thought to reduce inflammation, which is thought to be the root cause of many chronic illnesses
Polyphenols are plant-based compounds known for their antioxidant properties, which can promote health and offer protection against a range of diseases These compounds can be categorized into several groups, including flavonoids, phenolic acids, polyphenolic amides, and other types of polyphenols.
Polyphenols are beneficial compounds that may aid in preventing blood clots, lowering blood sugar levels, and reducing the risk of heart disease Additionally, they have the potential to enhance brain function, improve digestion, and provide some protective effects against cancer, although further research is necessary to confirm these benefits.
Overview of antioxidant activity
There is an increasing interest in plant-based secondary antioxidants as alternative sources to mitigate the harmful effects of existing antioxidant defense systems Both anaerobic and aerobic organisms maintain a delicate balance between the advantages and risks associated with oxygen utilization for energy During respiration, these organisms produce reactive intermediate compounds, including free radicals like superoxide anion (O2-) and hydroxyl radicals (OH-), as well as reactive oxidizing agents such as hydrogen peroxide (H2O2) These substances and their reaction products can lead to the destruction of essential biological molecules, including DNA, lipids, and proteins.
The body has developed enzyme systems, including superoxide dismutase, catalase, and glutathione peroxidase, to regulate highly reactive oxygen species (ROS) like superoxide and hydrogen peroxide, which are essential for detoxification However, environmental factors such as pollution, cigarette smoke, and ultraviolet radiation can overwhelm these protective systems, leading to increased ROS levels that contribute to disease and accelerate aging Plant-derived antioxidants, including carotenoids, flavonoids, and vitamins C and E, play a crucial role in supporting the body's defense against unwanted oxidation Ongoing research continues to explore the levels and benefits of these plant-based compounds.
2.5.2 Method for determination of antioxidant activity
There are various methods available to assess antioxidant activity, including Oxygen Radical Absorbance Capacity (ORAC), Total Radical-Trapping Antioxidant Parameter (TRAP), and 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) Among these, DPPH is preferred due to its distinct advantages over other testing methods.
- The method is simple, the required equipment is not too complicated
- Suitable for many antioxidant agents
Marsden Blois pioneered the DPPH method nearly 50 years ago in 1958, where he first investigated the antioxidant activity of the amino acid cysteine This was achieved by titrating cysteine with DPPH and measuring its absorbance over time at 515 nm.
The scientific name of DPPH is 1,1 diphenyl -2-picrylhydrazyl, the molecule is not dimerized like some other free radicals
DPPH is a stable free radical characterized by its purple solution, with an absorption maximum at 515 nm Antioxidants neutralize the DPPH radical by donating hydrogen, which reduces the absorbance at this wavelength and causes the solution's color to change from purple to light yellow.
Reaction mechanism of DPPH free radicals and antioxidants:
The antioxidant activity of a substance was determined by UV spectrometry using the reagent DPPH
The reaction utilizes the principle that DPPH generates stable free radicals in a saturated methanol solution When test substances are introduced, those capable of neutralizing or encapsulating free radicals will decrease the light absorption intensity of DPPH Antioxidant activity is assessed by comparing the light absorption values of the experimental solution to a control, measured at 515 nm using a colorimeter.
MATERIAL, CONTENTS, AND METHODS
Material
Medicinal origin: The medicinal herbs used in the experiment are lemongrass, cinnamon, and anise The medicinal herbs were purchased at Binh
An traditional medicine company (Nghia Trai village, Van Lam district, Hung Yen province) Medicinal herbs are dried, dried and ground into a fine powder
The study was conducted on strains of bacteria :
The bacteria in question are extensively characterized and included in the ATCC (American Type Culture Collection, USA), which houses the world's largest collection of microbial strains, viruses, animal and plant cells, and recombinant DNA This collection is supported by the Department of Internal Medicine - Diagnosis - Pharmacology - Toxicology and the Department of Veterinary Medicine.
3.1.3 Time and place of study
- Implementation period: From October 2022 to May 2022
Laboratory of Internal Medicine - Diagnosis - Pharmacology - Toxicology, Faculty of Veterinary Medicine, Vietnam National University of Agriculture.
Research contents
The study involved extracting medicinal compounds from lemongrass, cinnamon, and anise using six different solvents: Distilled Water (DW), Ethanol, Methanol, Hexane, Acetone, and Ethyl Acetate The resulting extracts were then diluted with DMSO to evaluate their biological activity in subsequent experiments.
- Investigation of antibacterial activity of medicinal herbs on 9 types of bacteria The extracts, in turn, were active at concentrations of 2000 mg/ml;
1000 mg/ml; 500 mg/ml and 250 mg/ml Using the results of measuring the diameter of the sterile ring to evaluate the antibacterial ability of medicinal herbs
- Extracting essential oils of medicinal herbs lemongrass, cinnamon, and anise by steam distillation
This study investigates the antibacterial activity of essential oils derived from medicinal herbs against nine types of bacteria using steam and diffusion methods The essential oils were tested at various concentrations: pure, 1/5, 1/10, 1/20, 1/40, and 1/80 The antibacterial efficacy of these essential oils was evaluated by measuring the diameter of the inhibition zones.
- Investigation of antibacterial activity of trans main standard cinnamon
The determination of polyphenol content in medicinal herbs involves diluting six extracts to a concentration of 20 mg/ml to measure the optical density at 750 nm, using chlorogenic acid standards The concentration of the extract is adjusted based on the color of the standard By establishing a correlation between the optical density values and chlorogenic acid, the polyphenol content of the extract can be accurately calculated.
The antioxidant activity of medicinal herbs was determined by adjusting the concentration of extracts to 1 mg/ml, 5 mg/ml, 20 mg/ml, and 40 mg/ml, based on the standard color of the VTME standard.
100mg/ml to measure the optical density value, establish the correlation between optical density gain value and VTME content Antioxidant activity was converted to VTME content.
Research Methods
3.3.1 Method of extracting medicinal herbs
Method of extracting medicinal herbs
Dried medicinal herbs were finely ground to a powder of less than 0.05mm and stored in plastic bags This medicinal powder was subsequently extracted using six polar solvents frequently utilized in plant extraction: ethanol, methanol, distilled water, ethyl acetate, acetone, and hexane.
The extraction process utilized organic solvents by soaking medicinal powder at room temperature for 24 hours, maintaining a ratio of 1:30, which means 10 g of medicinal herbs were combined with 300 ml of solvent.
With distilled water solvent, medicinal herbs are hot extracted by mixing the herbal powder with boiled distilled water at the ratio of 1/30 and stirrer for
To enhance the solubility of medicinal ingredients in plants, it is essential to avoid soaking in cold distilled water at room temperature Instead, traditional medicine often employs the decoction method, which involves heating the mixture for several hours or combining it with boiled water to create a tea This approach significantly improves the extraction of active compounds into the water.
The medicinal and solvent suspensions were then centrifuged at 3500 rpm for 10 min and filtered through a No2 qualitative paper to ensure the removal of any residue
The extract was evaporated under vacuum at low pressure to eliminate all solvents, ensuring the temperature remained at or below 40°C to preserve the biological activities of the medicinal herbs.
In bacterial experiments, the extracts obtained after evaporation are re-dissolved in dimethyl sulfoxide (DMSO) at a ratio of 10g of dry medicinal herbs to 5ml of DMSO This calculation is based on the original mass of the raw medicinal powder to ensure accurate content measurement, as the mass of the extract is typically minimal.
The extract will be further diluted to the next test concentrations using DMSO solvent, which is preferred over organic solvents like ethanol, methanol, and ethyl acetate due to its non-toxic nature and lack of impact on bacterial growth This ensures that any observed inhibitory effects on bacterial growth can be attributed to the active ingredients in the medicinal herbs The concentration system of the extract tested includes:
2000 mg/ml; 1000 mg/ml; 500 mg/ml and 250 mg/ml
Figure 3.1 Steps to perform the method of extracting medicinal herbs
Method of extracting medicinal essential oils
Step 1: Medicinal herbs are ground into small pieces, and soaked in 30% NaCl solution for about 12 hours
Step 2: After soaking, the medicinal herbs are steam-distilled During the distillation process, care should be taken to adjust the condensate temperature so that it is in the range of 30 - 40℃ (by adjusting the cooling water speed) because if the condensate is too hot, it will increase the solubility of the essential oil into the water and evaporate the essential oil
Figure 3.2 Essential oil distillation kit
Step 3: The mixture of essential oil and water is put into the separator After separation, we get crude oil and distilled water When put into the separator, the essential oils ligh ter than water will float to the top
Step 4 : Essential oil after water separation is divided into tubes to preserve
Figure 3.3 Extraction flask (Essential oils are lighter than water, so they separate large and float to the top when placed in the separator)
Figure 3.4 Essential oils of cinnamon, anise, lemongrass
3.3.2 Method of determining the antibacterial activity of medicinal herbs
The ability to inhibit bacterial growth was determined by the Kirby-Bauer agar diffusion method
Method to determine the antibacterial activity of herbal extracts
Figure 3.5 Describe the method of mixing bacteria into agar and punching a small well of antibacterial agent on agar plates
The Kirby-Bauer agar diffusion method was employed to assess the inhibition of bacterial growth, utilizing Miller Hinton Agar from Merck, Germany A total of 324 experiments were conducted, with each experiment repeated twice to ensure accuracy.
Utilize a specialized tool to chisel copper agar with a 1 cm inner diameter, creating four wells spaced 2-3 cm apart After introducing agar mixed with bacteria at a concentration of 10^6 bacterial cells/ml into the cage plate, apply the 4-well chisel to the agar surface Subsequently, add 100 µl of extract at varying concentrations to each well.
After being infused with medicinal herbs, the plates should be refrigerated for 3-4 hours to enable the extract to diffuse onto the agar surface (Bui Thi Tho and Nguyen Thi Thanh Ha, 2009).
After 24 hours of incubation at 37°C, agar plates were examined to evaluate the impact of bacterial growth inhibition The effectiveness of the inhibition was determined by measuring the diameter of the inhibition zone surrounding the well, utilizing a specialized ruler for accurate assessment.
Figure 3.6 The ability to inhibit bacteria of medicinal herbs within 24 hours of culture
Determination of the inhibition zone of medicinal herbs
To measure the diameter of the inhibition zone, use a pen and ruler to mark the diameter on the agar plate Position the plate so that the diameter line aligns with the center line of the screen set to zero The distance between the upper and lower edges of the inhibition zone, aligned with the screen line, represents the diameter of the inhibition zone.
D is the diameter of the inhibition zone of bacteria collected on the agar plate d is the diameter of the well (10mm)
Method to determine the antibacterial activity of essential oils
Determination of the antibacterial effect of essential oils based on steam method
The antibacterial activity of essential oil vapors was carried out according to the method of Edwards-Jonesa et al., (2004)
New colonies grown for 24 h on Muller Hinton agar were added to Muller Hinton gravy and adjusted to a concentration of 9x108 cfu/ml, by comparison
Cinnamon Hexane – ATCC 85922 was prepared using a standard MacFarland 3.0 solution, consisting of 0.3 ml of BaCl2 1% mixed with 9.7 ml of H2SO4 1% To achieve a final bacterial concentration of 10^6 cfu/ml for the antibacterial activity investigation, 25 ml of sterilized Muller Hinton liquid agar was cooled to 45 °C and inoculated with 27.8 µl of 9x10^8 cfu/ml bacteria before being poured into a 10 cm diameter petri dish Filter paper discs, 10 mm in diameter, were placed in the center of the agar plate, and 100 µl of various essential oils were added, while DMSO solvent was used for the control plate The agar plates were incubated at 37 °C for 24 hours, after which the bacterial inhibition levels of the essential oils were assessed Inhibition by fumigation was evaluated by measuring the diameter of the sterile ring where bacterial growth was prevented due to the essential oil vapors.
Figure 3.7 Antibacteria of essential oil based on steam method
Volatile oils demonstrate the capacity to inhibit bacterial growth, as evidenced by the diameter of the inhibition zone where bacteria are unable to thrive due to the effects of essential oil vapors.
Determination of the antibacterial effect of essential oils based on steam method
RESULTS AND DISCUSSION
The results of the investigation of antibacterial activity of medicinal herbs
This study explores the antibacterial properties of medicinal herbs such as lemongrass, cinnamon, and anise against nine different bacterial strains We evaluated the effectiveness of six selected solvents: hot water, methanol, ethanol, ethyl acetate, acetone, and hexane.
After 24 h of culture, the extracts of lemongrass, cinnamon and anise created inhibition zone of different sizes Some extracts produce a inhibition zone when acting on one bacterium, but not on other bacteria with a inhibition zone diameter The antibacterial ability on the same bacteria of different extracts is also not the same Therefore, it can be affirmed that the antibacterial activity of the extract depends on factors including: type of medicinal plant, concentration and extraction solvent
4.1.1 The results of determining the diameter of the inhibition zone of 6 extracts of lemongrass
Lemongrass was extracted using six different solvents: distilled water, methanol, ethanol, ethyl acetate, acetone, and hexane The antibacterial activity of these extracts was evaluated against nine types of bacteria by measuring the diameter of the inhibition zones produced.
The results on the antibacterial ability of lemongrass extracts of 6 solvents on 9 types of bacteria
After 24 h of culture, the extracts of lemongrass 6 solvents did not produce an inhibition zone on the bacteria or produced inhibition zone of different sizes The results are gathered in Table 4.1 and Table 4.2
Table 4.1 Results of inhibition zone diameter (mm) of lemongrass extracts
Table 4.2 Results of inhibition zone diameter (mm) of lemongrass extracts
From the results of Table 4.1 and Table 4.2, we confirm that the antibacterial activity of lemongrass extract was good against the bacteria group
B.subtilis ATCC 7953, S aureus ATCC 25023, S aureus ATCC 25923 and G phillus ATCC 7953 In which the largest inhibition zone was measured for the ethanol lemongrass extract against S.aureus ATCC 25023 (21.19 mm at the concentration of 2g/ml), and the solvent had good antibacterial activity against lemongrass with ethanol solvent Opposite, they are not well expressed with
DW, Ethyl, Hexane and Acetone solvents
4.1.2 The results of determining the diameter of the inhibition zone of 6 extracts of cinnamon
After 24 h of culture, the cinnamon extracts with 6 solvents did not produce inhibition zone on the bacteria or produced inhibition zone with different sizes The results are gathered in Table 4.3 and table 4.4
Table 4.3 Results of inhibition zone diameter (mm) of cinnamon extract
Table 4.4 Results of inhibition zone diameter (mm) of cinnamon extracts
The antibacterial activity of cinnamon extract was evaluated using six different solvents, with ethanol demonstrating the highest sterile ring diameter Notably, the strongest antibacterial effect was observed against S aureus ATCC 25023, achieving a diameter of 47.07 mm at a concentration of 2 g/ml These findings align with the earlier research conducted by Sen and Batra.
Research by Do et al (2014) and others has shown that ethanol extracts from certain medicinal herbs exhibit greater antibacterial activity compared to methanol and distilled water (DW) extracts Additionally, studies by Aladea P.I and Irobib O.N (1993) and Hemalatha et al (2011) confirm that ethanol extracts outperform methanol, chloroform, DW, acetone, ethyl acetate, and hexane extracts in terms of antibacterial efficacy in various medicinal plants.
4.1.3 The results of determining the diameter of the inhibition zone of 6 extracts of anise
After 24 h of culture, the anise extracts with 6 solvents did not produce inhibition zone on the bacteria or produced inhibition zone with different sizes The results are gathered in Table 4.5 and table 4.6
Table 4.5 Results of inhibition zone diameter (mm) of anise extracts
Table 4.6 Results of inhibition zone diameter (mm) of anise extracts
From 4.5 and 4.6, we confirm that, when comparing the antibacterial activity of anise extract using 6 solvents, methanol solvent gave the highest inhibition zone diameter of all bacteria In which, the strongest antibacterial activity was followed by B.subtilis ATCC 7953 (26.96mm at 2g/ml concentration) With ethanol and methanol extracts, inhibition zone were produced in all bacteria with varying degrees of magnitude Therefore, the antibacterial activity of the ethanol anise extract gave the best results on both Gram (+) and Gram (-) bacteria.
Results of the investigation on the antibacterial activity of essential oils
Antibacterial activity of lemongrass essential oil based on steam method Table 4.7 Antibacterial activity of lemongrass essential oil based on steam method (mm)
From the table 4.7, we confirm antibacterial activity of lemongrass essential oil based on the steam method, it has antibacterial activity on bacterial strains B.Subtilis ATCC 7953, S.aureus ATCC 25023, S.aureus ATCC 25923
Lemongrass essential oil exhibits strong antibacterial activity, particularly against B subtilis ATCC 7953, with a significant sterile ring diameter of 52.1 mm at pure concentration Additionally, it demonstrates antibacterial effects on S aureus strains at a 1/10 dilution The antibacterial efficacy of lemongrass essential oil, extracted using the steam method, is concentration-dependent, as indicated by a decrease in the diameter of the sterile ring with lower concentrations.
B.Subtilis strain at decreasing concentration)
Results of antibacterial activity of lemongrass essential oil based on diffusion method
Table 4.8 Antibacterial activity of lemongrass essential oil based on diffusion method (mm)
The antibacterial activity of lemongrass essential oil varies significantly between the steaming and diffusion methods, as shown in tables 4.7 and 4.8 While the steam method yielded different results, the diffusion method demonstrated antibacterial effects not only against B subtilis and S aureus but also against E coli strains ATCC25922 and ATCC85922.
4.2.2 Results of antibacterial activity of cinnamon essential oil
Antibacterial activity of cinnamon essential oil based on steam method Table 4.9 Antibacterial activity of cinnamon essential oil based on steam method (mm)
Bacteria Concentration of essential oil
Cinnamon essential oil exhibits significant antibacterial activity, particularly against the S aureus strain, as confirmed by the results in Table 4.9 The effectiveness of this essential oil is concentration-dependent, with a 20% dilution yielding the largest sterile ring diameter of 29.82 mm for S aureus ATCC 25023 and 29.86 mm for S aureus ATCC.
Antibacterial activity of cinnamon essential oil based on diffusion method
Table 4.10 Antibacterial activity of cinnamon essential oil based on diffusion method (mm)
Table 4.10 demonstrates that cinnamon essential oil exhibits antibacterial activity against all nine bacterial strains tested using the steam method, with the most potent effect observed against one specific strain.
S.aureus (diameter of inhibition zone is 33.21 with S.aureus ATCC 25023 and 33.92 with S.aureus ATCC 25923) The antibacterial activity of cinnamon essential oil is influenced by the concentration of the essential oil, in particular, the antibacterial activity of the essential oil decreases with decreasing concentration
4.2.3 Results of antibacterial activity of anise essential oil
Antibacterial activity of anise essential oil based on steam method
Anise essential oil did not have antibacterial activity against 9 bacteria strains including E.coli ATCC 25922, E.Coli ATCC 85922, E.Coli ATCC 35218,
B.Subtilis ATCC 7953, G.Phillus ATCC 7953, P.Seudo ATCC 9027, S.aureus
ATCC 25023, S.aureus ATCC 25923, Salmonella ATCC 13311
Antibacterial activity of anise essential oil based on diffusion method Table 4.11 Antibacterial activity of anise essential oil based on diffusion method (mm)
From the table 4.11, we confirm that anise essential oil has weak antibacterial activity Antibacterial activity of anise essential oil was present on
S.aureus ATCC 25023 but to a small extent (diameter of inhibition zone is 13.22 mm)
4.3 The results of the investigation of the antibacterial activity of the main active ingredients in cinnamon
Cinnamon bark oil primarily consists of cinnamaldehyde (65-80%) and eugenol (5-10%), along with other significant compounds such as cinnamic acid, cinnamyl acetate, α-thujene, α-terpineol, α-cubebene, β-caryophyllene, and terpinolene This essential oil exhibits various biological activities, including antioxidant, antimicrobial, antifungal, and antidiabetic properties The thesis explores the botanical origin of cinnamon, the production of its essential oil, and the biological activities linked to both the essential oil and its individual components (Petter et al., 2018).
Trans-cinnamaldehyde constitutes 65% to 85% of cinnamon essential oil; however, even when present in 100% concentration, it does not exhibit a larger inhibition zone diameter against certain bacteria compared to cinnamon essential oil, as demonstrated in Table 4.13.
Table 4.12 The results of antibacterial activity of trans-cinnamalehyde with different concentrations (mm)
Reagent Bacteria Concentration of essential oil
The results of determining polyphenol contents
Table 4.12 confirms that trans-cinnamaldehyde exhibits inhibitory effects on all nine tested bacteria Additionally, cinnamon essential oil also inhibited all nine bacteria, showing larger inhibition zones This indicates that the antibacterial properties of cinnamon are not solely due to trans-cinnamaldehyde, but also involve other unidentified components present in cinnamon.
The findings highlight the necessity for further research to pinpoint the primary active compounds in cinnamon that contribute to its antibacterial properties, as these components are crucial in exerting pharmacological effects However, it is important to note that the medicinal properties of these plants are only partially effective against bacterial growth.
4.3 The results of determining polyphenol contents
4.3.1 The result of building a standard graph between chlorogenic acid content and the increase in optical density measured when reacting with
In our experiment to determine polyphenol content, we utilized chlorogenic acid as a standard for quantifying the polyphenol levels in medicinal herbs We established a correlation graph between chlorogenic acid concentration and the corresponding increase in optical density (OD) values, which were measured after reacting with Folin-Ciocalteu reagent The results were compiled in a table and illustrated in a graph.
Table 4.12 Variation of OD values according to chlorogenic acid standard concentration (mg/ml)
Blank 1 Blank 2 Optical density Medium SD SE
Figure 4.2 Correlation between content of chlorogenic acid standard substance (mg/ml) and the degree of increase in optical density value (OD value) y = 0,1367x - 0,029 R² = 0,9974
The study revealed a strong positive correlation between chlorogenic acid content and the increase in optical density, with a coefficient of determination of R² = 0.9974 and a p-value of less than 0.001 This correlation is utilized to quantify the polyphenol content in the experimental samples.
4.3.2 The results of polyphenol content of herbal extracts of cinnamon, anise, lemongrass (content 100mg/ml)
Table 4.13: Polyphenol content of medicinal herbs converted to chlorogenic acid (mg/100 mg of medicinal herbs) when extracted with different solvents
Polyphenol (mg chlorogenic acid/100mg medicine) Hot water Ethanol Methanol Ethyl acetate
Experimental results show that all the investigated medicinal herbs contain polyphenols with different concentrations, depending on the species and extraction solvent
To compare the polyphenol content of the three medicinal herbs, we compared the contents of the extracts extracted by the same solvent The results are shown in the Figure
Figure 4.3 Total polyphenol content converted to chlorogenic acid (mg) of cinnamon, anise, lemongrass (content 100mg/ml) when extracted with different solvents
The analysis of polyphenol content in various solvent extracts revealed that methanol, ethanol, distilled water (DW), acetone, and cinnamon extract yielded the highest levels, with methanol cinnamon extract reaching 42,996 mg of chlorogenic acid per 100g of medicinal herbs Anise and lemongrass followed closely in polyphenol content In contrast, ethyl acetate and hexane extracts showed that anise had a higher polyphenol content than other extracts Overall, methanol, ethanol, DW, and acetone solvents consistently produced higher polyphenol levels compared to hexane, which recorded the lowest content at 1,719 mg, 3,456 mg, and 1,298 mg of chlorogenic acid per 100g of the three medicinal herbs, respectively.
4.3.3 The results of polyphenol content of essential oils of cinnamon, anise, lemongrass (1ml content)
Table 4.14: Polyphenol content of medicinal herbs converted to chlorogenic acid (mg/ml pure essential oil)
Experimental results show that all the investigated medicinal herbs contain polyphenols with different concentrations, depending on the species and extraction solvent
To compare the polyphenol content of anise, cinnamon, and lemongrass, we compared the content The results are shown in the chart
Figure 4.4 Total polyphenol content converted to chlorogenic acid (mg) of
3 essential oils (converted to 1 ml of pure essential oil)
Lemongrass essential oil at a 1/10 concentration exhibited the highest polyphenol content, measuring 62.692 mg of chlorogenic acid per 100g of medicinal herbs, as shown in Table 4.14 and Figure 4.4 In comparison, anise and cinnamon followed in polyphenol content, with cinnamon showing the lowest level at 26.225 mg of chlorogenic acid per 100g at the same concentration.
4.4 Results of determining the antioxidant activity of herbal extracts and essential oils
4.4.1 The results of the determination of the antioxidant capacity of the standard VTME (Alpha tocopherol)
VTME (Alpha tocopherol) serves as a standard for measuring the antioxidant capacity of medicinal herbs A standard graph was created to correlate the concentration of VTME with the antioxidant capacity assessed using DPPH The results are presented in tables and figures.
Figure 4.5 The color cups represent the color change of DPPH solution induced by the antioxidant activity of VTME ) at different concentrations
(from right to right: Blank, VTM E 0.05mg/ml; 0.1 mg/ml; 0.15mg/ml;
0.2mg/ml; 0.25mg/ml; 0.3mg /ml; 0.4mg/ml; 0.45mg/ml; 0.5mg/ml)
Table 4.15 Antioxidant activity of standard VITAMIN E determined by
DPPH method at different concentrations AA%
Antioxidant activity content No.1 No.2 Concentration
Figure 4.6 Correlation between content of VTME standard (standard substance) and antioxidant activity (scavenging activity) y = 11,581x + 1,2322
The study reveals a significant positive correlation between VTME content and the increase in optical density when reacting with the DPPH reagent, indicated by a coefficient of determination R² of 11.581 and a p-value of less than 0.001 This correlation will facilitate the equivalent conversion of the optical density increase generated by the extracts in reaction with the DPPH reagent.
4.4.2 The results of the determination of antioxidant activity of extracts of cinnamon, anise, lemongrass
Figure 4.7 Color change of cinnamon at a concentration of 20mg/ml of medicinal herbs upon color reaction with DPPH
The color intensity of medicinal herbs when reacting with DPPH indicates their antioxidant index, with darker colors correlating to lower antioxidant activity, as illustrated in Figure 4.3 Specifically, hexane cinnamon and ethyl cinnamon in the 2nd and 3rd cuvettes exhibited antioxidant activities of 1,167 mg and 1,112 mg, respectively, at a concentration of 20 mg/ml Conversely, a yellower hue in the tested medicinal herbs signifies higher antioxidant activity, as demonstrated in Figure 4.4, which shows the color reaction of cinnamon herbs at a reduced concentration of 10 mg/ml.
Figure 4.8 Color change of cinnamon medicinal herbs at a concentration of
10mg/ml of medicinal herbs upon color reaction with DPPH
The antioxidant activity of medicinal herbs was assessed after adjusting the concentration of extracts, with the findings summarized in Table 4.18.
Table 4.16 Antioxidant activity of extracts
Antioxidant activity converted to VTME (mg)
DW Ethanol Methanol Ethyl Acetone Hexane
To compare the antioxidant activities of the three medicinal herbs, we compared the antioxidant activities of extracts extracted by the same solvent The results are shown in the figure 4.5
Figure 4.9 Total antioxidant activity of lemongrass, cinnamon and anise extracts was converted to VTME content (mg/100 mg of medicinal herbs)
The analysis of table 4.18 and chart 4.3 reveals that methanol, ethanol, distilled water (DW), acetone, and cinnamon extract exhibit the highest total antioxidant activity, with anise and lemongrass following closely In contrast, lemongrass extract demonstrated lower antioxidant activity in ethyl acetate and hexane solvents, with no activity detected in hexane Notably, the methanolic cinnamon extract contained the highest polyphenol content at 76,796 mg of chlorogenic acid per 100g of the medicinal herb Overall, methanol, ethanol, DW, and acetone solvents consistently yielded greater total antioxidant activity compared to ethyl acetate and hexane.
Extracts from lemongrass, cinnamon, and anise demonstrated significant antibacterial properties across three solvents: distilled water, methanol, and ethanol These extracts also exhibited high polyphenol content and antioxidant activity, aligning with previous research that links antibacterial efficacy to polyphenol levels and antioxidant capacity Notably, Nguyen Thi Thanh Ha et al (2021) highlighted that medicinal herbs with the ability to inhibit bacterial growth also possess elevated polyphenol content and robust antioxidant activity Additionally, Thanh Van Nguyen and Hai Thanh Nguyen (2019) established a positive correlation between the polyphenol content, antioxidant activity of medicinal herbs, and their antibacterial effectiveness.
4.4.3 Results of determining the antioxidant capacity of essential oils
Table 4.17 Antioxidant capacity of essential oils extracted
Essential oil Antioxidant activity converted to VTME (mg)
This study evaluates the antioxidant activity of three essential oils: lemongrass, anise, and cinnamon The comparative analysis of these essential oils reveals their respective antioxidant properties, as illustrated in the accompanying figure.
Figure 4.10 Total antioxidant activity of medicines essential oils converted to VTME content (mg/ml of pure essential oil)
Lemongrass essential oil at a concentration of 1/10 exhibited the highest antioxidant activity, measuring 41,143 mg of chlorogenic acid per 100g of medicinal herbs, as shown in Table 4.19 and Figure 4.4 In comparison, anise and cinnamon followed in antioxidant activity, with cinnamon showing the lowest at 10,163 mg of chlorogenic acid per 100g of medicinal herbs at the same concentration.
Our research highlights the antibacterial properties of cinnamon, anise, and lemongrass, supporting their traditional use in treating bacterial infections The antibacterial activity of these plant extracts is closely linked to their polyphenol content and antioxidant properties, indicating the significance of these compounds in combating bacteria Notably, cinnamon exhibited the strongest antibacterial effects, effectively inhibiting antibiotic-resistant strains such as E coli, S aureus, B subtilis, and G philus, positioning it as a potential alternative to conventional antibiotics Furthermore, the antibacterial and biological activities of these plants were more pronounced in their volatile fractions compared to non-volatile ones, underscoring the need for further research to fully assess their therapeutic potential.
1 Bui Thi Tho (2003), "Thuốc kháng sinh và nguyên tắc sử dụng trong chăn nuôi", NXB
2 Bui Thi Tho and Nguyen Thi Thanh Ha, Giáo trình dược liệu học thú y, 2009
3 Do Tat Loi, 1995 “Những cây thuốc và vị thuốc Việt Nam” Nhà xuất bản Khoa Học và Thuật Hà Nội