In vitro Evaluation of the Antimicrobial Effect of Common UAE Natural Herbals on Different Gram-positive and Gram-negative Bacteria

The main goal of this research is to assess the antibacterial effectiveness of commonly used UAE natural herbs, including Turmeric, Henna, Sidr


INTRODUCTION
To address antibiotic resistance, there has been an increasing demand to develop a novel antibiotic drug derived from diverse sources in recent years.Antibiotic resistance is a major problem nowadays, causing failures of treatment of diseases caused by microorganisms resistant to many drugs and raising concerns for world health.Therefore, the development of new antimicrobial remains a crucial objective, with natural products continuing to be a major source. 1 Herbs have existed from time immemorial and have been used for many uses, including for the treatment of many diseases.So, in our research, we dealt with some herbs to test if those herbs do effects different types of Gram-negative and positive bacteria.
Henna is a flowering plant that has derived from the henna tree or Lawsonia inermis also called the Egyptian privet.It's a woody plant with short to long size.The main color of the flower ranges from yellow to pink color which gives small fruit.But the powder reddish-brown dye color of henna that comes from its leaves.The origins of the henna tree are in Asia, Australia, and North Africa. 2 The plant's enormous amount of nutrients and chemicals may have anti-inflammatory, hypotensive, antibacterial, astringent, and antiviral effects, that can be used as a medicine to treat many diseases.In conventional medicine, they used roots, stem bark, seeds, and flowers of henna to treat burns, wounds, reduce fever, and others. 3urmeric also called Indian saffron, diferuloylmethane, or Curcumin in other languages.Derived from the Curcuma longa plant that belongs to the Zingiberaceae family.It's native to South Asia. 4 Turmeric is used for cooking spices and in the medical field turmeric have an anti-inflammatory and antioxidant effect, as for traditional medicine, they used the rhizome part for clinical purposes like arthritis, poisoned food or digestive disorder, cancers, cleaning wounds, skin problems, In Ayurvedic medicine (Indian medicine) they use turmeric to treat several respiratory conditions.But for now, they use it in the manufacture of sunscreen and dermal creams.Via boiling the herb-dried then grinding it to become a yellow powder that color comes from the polyphenolic compound. 5idr is a dense, deciduous, and spreading tree with a branching trunk of light brown zigzag branches, reaching a height of approximately 2 to 4 meters.Its leaves are small, elliptical, and have a thick scale. 6n desert, semi-desert, and coastal regions, the Sidr plant thrives.Its leaves, abundant in magnesium, iron and calcium, possess medicinal properties effective against various illnesses.Its powerful antimicrobial and anti-inflammatory attributes are utilized in crafting wound antiseptics, and its resin-derived oil is used in deodorants. 7yrrh herb is a mucilage-like substance secreted from a certain type of plant called (Commiphora myrrha).Traditionally, it has been used to address a range of ailments including injuries, buccal ulceration, gastric illness, bacterial diseases, and inflammation conditions.Its versatile properties encompass acting as mucolytic, emmenagogs, carminative, antiparasitic, astringent, and antibacterial.The phytochemical analyses revealed a spectrum of compounds within it, including terpenoids, for example, steroids, diterpenoids, triterpenoids, essential oil, and sesquiterpenoids.Additionally, aromatherapy, cosmetics, and perfumes all use its original oil.
Scientific investigation has underscored its multifaceted biological activities, which include anti-inflammatory, antioxidant, antimicrobial, anthelmintic in addition to its activity against diabetes and malignancy.Recently, research has even demonstrated its efficacy against pulmonary diseases such as coronavirus infections.
Considering the ongoing progress of medication production, there is hope that its diverse phytochemical constituents could be further explored.Potential avenues include its application against parasites.Additionally, deeper elucidation of its interactions with pharmaceutical drugs holds promise for optimizing therapeutic outcomes. 8ethanolic leaf extracts of plants, including T. procumbens, T. cordifolia, and C. igneus, had strong antibacterial action against a variety of Gram-positive and Gram-negative pathogens, according to research by Pallavi et al.Comparing these studied plant extracts to the common antibiotic ciprofloxacin utilized in this investigation, they showed increased efficacy. 9sing natural antibacterial substances from renewable resources improves the resilience and performance of textiles and considerably mitigates the ecological consequences conventionally linked to treatments with chemicals. 10

MATERIALS AND METHODS
Different Gram-negative and Grampositive organisms such as (Streptococcus agalactiae (group B streptococci), Streptococcus pyogenes (group A streptococci), S. aureus, E. coli, E. faecalis, P. aeruginosa, K. pneumoniae, and P. mirabilis), were enrolled in the in vitro analysis to assess the antimicrobial effect of the extracted herbal compounds.

Extraction of herbal
The plant materials used for determining antimicrobial activity were collected in different forms: Henna (Lawsonia inermis Linn) in powder form, Turmeric (Curcuma longa) in powder form, Sidr (Ziziphus Jujuba) in leaves form, and Myrrh in gum-resin form.
The collected plant parts underwent a series of steps for preparation.They were initially washed, disinfected, and cleaned with tap water, followed by air drying until ready for use.The herbal extraction method followed the protocol outlined by Hemeg et al.Distilled water served as the solvent for the dried plant material, whether in powder, leaf, or gum resin form.
To initiate the extraction process, 10 grams of the specified plant material were added to 100 mL D. W. in a sterile container.The solution allowed to soak for 24 hours at room temperature or in a cool, dry environment.After the designated extraction period, the residual plant material was separated from the solvent.Initially, rough clarification was achieved through decanting, followed by filtration using Whatman filter paper to obtain a clear filtrate.The resulting extract yields were then transferred into small sterile containers and stored in the refrigerator at temperatures between 2-4°C until required for further use. 11

Preparation of organisms
Eight organisms were used to assess each herbal extract's antimicrobial efficacy.Four organisms were Gram-positive (group A streptococci, group B streptococci, Enterococcus faecalis and S. aureus) and four organisms were Gram-negative (Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae).All organisms were obtained and well-identified by subculture.In the sub-cultivation of bacteria, a loop was used to capture a bacterial colony, then used to inoculate into fresh nutrient agar and blood agar (line for isolation), then placed in the incubator at 37°C overnight (18-24 hours) aerobically and then refrigerator (2-4°C).

Antibacterial activity
Serial dilution is employed to prepare three concentrations of each extract: 100%, 75%, and 50%.The total volume for each extract concentration was 1000 mL.
The inoculum suspension was prepared using pure colonies of each microorganism from agar plates.In the agar diffusion assay, the bacteria suspension was spread evenly across the entire Mueller Hinton agar plate surface.Three wells were aseptically punched into the agar, and each well received (100 µL) of the herbal extract at different concentration corresponding to the 50%, 75%, and 100% diluted extracts.The inoculated plates were then incubated aerobically at 37°C overnight.The herbal extract diffused into the agar, inhibiting the growth of the test organisms used.
Antimicrobial activity was assessed by measuring the diameter of the inhibition zone in millimeters.

RESULTS
The antibacterial efficacy of herbal extracts, determined through the agar well diffusion method, varied between 10 mm and 36 mm in diameter.
Among the four herbal extracts, Henna showed the largest zone of inhibitions (36 mm), exhibiting sensitivity to Staphylococcus aureus, Group A Streptococci, Group B Streptococci, and Klebsiella pneumoniae.In Figure 1, the zones of inhibition at concentrations of 100%, 75%, and 50% were 35 mm, 30 mm, and 30 mm, Figure 5. Shows that the largest zone of inhibition of Group A Streptococci is observed with Henna, followed by Myrrh and Turmeric, with no inhibition seen with Sidr Figure 6.Shows that the largest zone of inhibition of Group B Streptococci is observed with Sidr, followed by Henna and Myrrh, with no inhibition seen with Turmeric respectively, against Staphylococcus aureus.For Group A Streptococcus (Streptococcus Pyogenes), the diameters were 36 mm, 35 mm, 32 mm, while for Group B Streptococcus (Streptococcus agalactiae), they were 17 mm, 15 mm, 14 mm.Against Klebsiella pneumoniae, the diameters were 12 mm, 10 mm at concentrations of 100% and 75%.
Myrrh showed antibacterial effect against all bacteria, the range of the size of the zone of inhibition varied between 11 and 16 mm, except Escherichia coli at a concentration of 50%, show no effect (Table ).
Sidr displayed inhibitory effects against Staphylococcus aureus (20 mm at all concentrations) and Group B Streptococcus (Streptococcus agalactiae) with diameters of 22 mm, 20 mm, 19 mm at concentrations of 100%, 75%, and 50%.Escherichia coli showed sensitivity only at a concentration of 100%, with a 12 mm diameter (Figure 2).Turmeric exhibited a comparatively lower antibacterial effect.Against Group A Streptococcus (Streptococcus Pyogenes), the diameters were 12 mm, 12 mm, 10 mm.For Proteus mirabilis, they were 14 mm, 14 mm, 13 mm, and against Escherichia coli, only at a concentration of 100%, a 12 mm diameter was observed (Figure 3).
The antibacterial activity evaluation of these plant extracts is summarized in Table and charts represented in Figures 4-11.

DISCUSSION
Herbs exhibit promising efficacy against diseases and their causative agents, prompting research into the efficacy of UAE herbs against various bacteria.Our investigation revealed that Henna, Sidr, Myrrh, and Turmeric, extracted via water extraction method, exhibit varying reactions against different Gram-negative and Gram-positive organisms.
A study conducted in Iraq by Kathem K. Al-Rubiay in 2008, demonstrated that the  oil and alcohol extraction of Henna displayed antimicrobial activity against Staph aureus, Staphylococcus epidermidis, P. aeruginosa, and -hemolytic streptococci.While our study corroborated its impact against Gram-positive bacteria, it did not yield positive results against Gram-negative bacteria. 12urmeric exhibited potent antibacterial effects in our study against Gram-positive bacteria such as Group A Streptococcus, and Gramnegative organisms including Proteus mirabilis and E. coli, consistent with findings from a study at the Microbiology Department, Quaid E Azzam University, Pakistan. 13urthermore, research by Noha Khalil at the college of Pharmaceutical Sciences and Pharmaceutical Industries, Future University, New Cairo, revealed that the oil extract of Myrrh effectively targeted S. aureus, K. pneumoniae, P. aeruginosa, and E. coli.In comparison, our study demonstrated efficacy against additional bacteria including Streptococcus pyogenes, Streptococcus agalactiae, and Enterococcus faecalis.

Figure 1 .
Figure 1.Illustrates the zone of inhibition, which indicates the sensitivity of Group A Streptococci, S. aureus, Group B Streptococci, and Klebsiella pneumoniae to Henna at 100% and 75% concentrations.In contrast, Enterococcus faecalis, Proteus mirabilis, Escherichia coli, and Pseudomonas aeruginosa exhibit resistance

Figure 4 .
Figure 4. Shows that the largest zone of inhibition of Staphylococcus aureus is observed with Henna, followed by Sidr and Myrrh, with no inhibition seen with Turmeric

Figure 7 .
Figure 7. Shows that the zone of inhibition of Enterococcus faecalis is only observed with Myrrh, and no inhibition seen with other herbs

Figure 8 .
Figure 8.The graph indicates that Myrrh is the most effective herbal extract against E. coli, followed by Turmeric and Sidr, while Henna shows no antibacterial activity

Figure 9 .
Figure 9.The graph indicates that Myrrh is the most potent herbal extract against K. pneumoniae, followed by Henna, while Turmeric and Sidr show no antibacterial activity

Figure 10 .
Figure 10.The graph clearly indicates that Myrrh is the only effective herbal extract against P. aeruginosa, while Turmeric, Sidr, and Henna show negligible antibacterial activity

Figure 11 .
Figure 11.The graph indicates that Myrrh is the most potent herbal extract against Proteus mirabilis, followed by Turmeric, while Henna and Sidr show no antibacterial activity

Table .
Illustrates the correlation between the inhibition zones of test organisms and different concentrations of the herbal extracts.The results indicate that the effectiveness of the herbal extracts varies significantly depending on the type of bacteria and concentration used.Henna and Myrrh exhibit broader antibacterial properties compared to Turmeric and Sidr Correlation of inhibition zone and type of Herbals