Evaluation of Antioxidants, Antibacterial and Antidiabetic Activities of Aqua-alcoholic Marjoram Extract

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Introduction
Recently, several studies about plant extracts were conducted that included their properties in nano size, antioxidant [1][2][3][4] , their phenolic content , and their cytotoxicity due their bio-activities and safety [5][6][7] , in addition to other studies that concerned with diabetes complications [8][9][10][11] .Marjoram is an aromatic herbaceous plant and it is from the famous mint group and contains many vitamins and minerals, as well as volatile oils, flavonoids, hydroquinone glycosides, sugars, and triterpenes.These ingredients give Marjoram important health properties such as antimicrobial, anti-inflammatory, analgesic, antidepressant, and antiviral.Marjoram has been employed for the treatment of a wide range of diseases as a traditional medicine including disorders of the heart, lungs, gastric and nerves 12 .Plant extracts represent the major source of the antioxidants compounds due to the presence of the active compounds like phenolic acids and flavonoids 13 .These types of phytochemical have the ability to scavenger free radicals and protect cells from damage by reducing oxidative stress 14 .More recently, natural products are choosing for synthetic additives.Generally, plant extracts are used as an important source of phytochemicals that possess multi-biological activities including antioxidants, antiviral and antimicrobial activities 15 .Oxidative stress considers one of the main reasons for the initiation and progress of diabetes mellitus, heart and cancer diseases due to generate reactive oxygen and nitrogen species, which represent unstable molecules occurred endogenously through aerobic metabolic processes 16 .
Carotenoids and tocopherol consider natural antioxidants that obtained from plant extracts, in addition to other important secondary metabolites like kaempferol and quercetin that were found to be as excellent antioxidants 17 .More recently, medicinal plant extracts showed a high rank of antioxidants activity toward degenerative disease that reached to 75% in terms of the inhibition percentage of oxidants.Furthermore, it was found that no significant effect of the extraction solvents on the obtained plant extract yield 18. In another recent study, it was reported that that the extracts of medicinal plants act as vital antioxidants in treatment of wide range of degenerative diseases and inhibition oxidants to avoid the oxidative stress 19 .
One of the major public health problems is the acquisition of bacterial resistance to traditional antibiotics, which is required a continuous search for new antibiotics that work properly and safety as effective antibiotics 20 .Due to this problem, plant extracts have received great attention because they contain biologically active and non-toxic components to study the extent of their effect on bacterial and fungal cells 21 .
It was reported that vegetal sources are used in development in many drugs, because plant extracts possess important properties like antiinflammatory, antibacterial and antioxidant effects.These properties can be employed in development of new drugs with no side effects 22 .More recently, several plant extract tests showed high action against selected pathogenic bacteria.Antibacterial activities can be attributed to presence bioactive compounds that support the antibacterial activity 23 .Antibacterial activities were evaluating in plant extracts.It was found that these activities are associated with certain phytochemical components.These components include tannins, saponins, flavonoids, alkaloids.They are support the activities against bacteria, diabetes, oxidants and cancer 24,25 .
Diabetes is considered one of the diseases that is a major challenge to public health, and it represents a group of metabolic disorders that are characterized by hyperglycemia, beta-cell impairment in insulin secretion, insulin resistance, or both.Moreover, diabetic complications could be associated with variation in the body's antioxidant defense system, oxidative stress, and dyslipidemia

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. Studies reported that plant extracts that contain the active components like isoflavones possess hypoglycemic activity.Furthermore, it was found that the presence of alkaloids, phenolic compounds, flavonoids, and terpenoids leads to lowering the glucose level in diabetic patients 27 .
More recently, antidiabetic activity of plant water crude extracts has been investigated.The study referred to that glucose level was decreased regularly with increase treatment time and the rate of glucose decreasing was increased with increasing of the applied dose 28 .Antidiabetic activity was detected in another plant extracts with different extraction solvents.The study confirmed that the plant extract of honeybush tea displayed antidiabetic and antioxidant activities.Furthermore, the study reported that this type of plant extracts considers a promising therapeutic agent in avoidance and controlling on diabetes and its complications 29 .The extract of Chromolaena odorata was used to treat diabetes and several infection diseases.The results of this study support the ability of this type of plant extract to be as promising antidiabetic agent and some infection disease 30 .
The aim of this study is to determine the bio activities of marjoram extracts toward diabetes in terms of certain relevant biochemical factors including glucose, insulin, liver functions, lipid profile and testosterone via treatment diabetic mice group related to control group and its activities toward pathogenic bacteria and oxidants.https://doi.org/10.21123/bsj.2024.9328P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal

Extraction of Marjoram Leaves
Powder of dried leaves of marjoram (10 gm) was extracted by 70% methanol to obtain the aqua methanolic extract depending on the ratio 1:10 (weight: volume) at 60-80 Cº for 6-8 hrs.The process was conducted by soxhlet.The result of the extraction process was filtered by a filter paper type Whatman No. 1, then the filtrate dried at 40 Cº and stored at -21 Cº, then qualitative tests for alkaloids, terpenes, and flavonoids were conducted depending on Mujeeb F, et al 31 .Also, the antioxidant activity of the studied extract was conducted depending on the DPPH method which was reported elsewhere 32 .The antibacterial activity test was achieved by a plate agar method 33 .The toxicity of marjoram extract was studied by applying the LD50 procedure 34 .The antidiabetic activity was assessed in terms of many biochemical factors including glucose, insulin, liver functions, and lipid profile in alloxandiabetic mice.The mentioned biochemical parameters were evaluated by their suitable kits by spectrophotometric methods.Fertility improvement in the treatment mice group was evaluated in terms of testosterone level in both diabetic and control groups.

Antidiabetic Activity
The antidiabetic activity was estimated depending on certain animal model design.The model includes diabetes induction by alloxan in a group of mice and then the diabetic mice were treated with marjoram extract with a certain dose and period related to positive and negative control groups, as reported below.

Animals Care
Thirty-two healthy mice were employed in the current study.The animals were purchased from the Center of Biotechnology, University of Al-Nahrain.The age of animals is three months and the weight of each mouse is about 25 grams.Cages of mice were cleaned with soap and tap water together with 70% ethanol for sterilization.Furthermore, mice have been exposed to light at a room temperature, 25°C for 12 hrs.

Diabetes Induction
Diabetes was induced in mice by injecting (IP) with alloxan monohydrate after mixed with saline in a ratio 5% w/v and volume 0.1 ml and at dose (150mg/kg BW).The diabetic condition was formed after 48 hrs., of injection with alloxan and was confirmed by weight loss and hyperglycemia.

Mice groups
In the case of the anti-diabetic effect by Marjoram extract in the current study, thirty-two mice were employed to achieve this part of this study.The animals were divided into four groups which are classified as follows.Group 1: normal mice (normal control group).Group 2: normal mice injected with alloxan (diabetic group, positive control group).Group 3: diabetic mice treated with 0.5 ml of marjoram extract solution containing 4mg of extract (treated group).Group 4: normal mice treated with the same dose of extract used in group 3 (negative control group).

Blood Samples Collection
Samples of blood were collected by heart puncture occasionally at the period of the treatment by Marjoram extract.The mice were killed by cervical dislocation after they fasted overnight.

Measurements of Biochemical Parameters
Agappe diagnostic kits were used to estimate all the studied biochemical parameters in the current study.Level of fasting blood glucose, lipid profile, testosterone and enzymes of AST and ALT were determined according to kits procedures and spectrophotometric methods.

Statistical Analysis
Data for all the studied parameters in the current study were stated by mean SD.Statistics data were estimated statistically by analysis of variance [ANOVA] using SPSS program version 17.The significant differences were taken when P 0.05.https://doi.org/10.21123/bsj.2024.9328P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal

Qualitative Tests
The brownish-red precipitate was the result after adding 1%HCl and few drops of Dragendorff reagent to the filtered boiled methanolic extract.This colored precipitate indicates the presence of an alkaloid.The presence e of saponins was confirmed after a vigorous shake for a few minutes of aqua extract to form a stable foam.A blue-black precipitate referred to the presence of tannins in this extract.The precipitate formed from the addition of FeCl 3 reagent (few drops) to the aqueous extract.Flavonoid was indicated by a yellow coloration formed from the addition of concentrated H 2 SO 4 to the mixture of aqua extract and ammonia solution.A reddish brown color confirmed the existence of the active component terpenoids in the studied extract when a chloroform extract was added to the concentration of sulphuric acid carefully, as shown in Table 1.

Determination of the Half Lethal Dose (LD50)
The results of LD50 have been recorded in Table 2. Fig. 1 shows that the value of LD50 was found to be 80.47 mg/kg BW, also the allowed dose was found to be 12.5 mg/kg that means for a person with 70 kg body weight the allowed dose of this extract is equal to 875 mg/70 kg BW, (0.875 gm/70 kg BW).

Antibacterial Antifungal Activities
Table 3 shows the activity of Marjoram extract toward selective pathogenic bacteria and fungi.Four different doses including 4mg, 2mg, 1mg, and 0.5mg were applied as allowed doses according to LD50 results.Pathogenic bacteria include Eshreshia, coli, Pseudomonas aeruginosa, Klebsiella, Acinetobacter baumannii, as gram negative bacteria species, and Staphylococcus aureus, Streptococcus pyogenes, Staphylococcus hominis, Staphylococcus epidermidis, Enterococcus Spp., as gram positive bacteria species together with Candid albicans and Candida tropicalis as pathogenic fungi.The results revealed that marjoram extract possesses high activity toward each Pseudomonas aeruginosa, Streptococcus pyogenes, Staphylococcus epidermidis, Enterococcus Spp., Acinetobacter baumannii, Candid albicans and Candida tropicalis while both Klebsiella and Staphylococcus hominis were affected by extract at the first upper concentration and first and second upper concentrations, respectively, but there is no effect of the extract on E. coli and Staphylococcus aureus even at high concentrations (doses), as shown in Table 3.

Antioxidant Activity
Four different concentrations of Marjoram extract were applied to evaluate the extract's antioxidant activity, they are including 25μg/ml, 50μg/ml, 100μg/ml, and 200μg/ml.ascorbic acid was used as reference antioxidant to determine the extract's antioxidant activity.The results of the Marjoram extract were recorded in Table 4.They showed high antioxidant activity in the concentrations 200,100 and 50 μg/ml, but no 25 μg/ml, there is a significant difference between extract antioxidant activity and antioxidant activity of ascorbic acid at this concentration (25μg/ml), whereas, no significant differences between ascorbic acid antioxidant activities and extract antioxidant activities at the concentrations 200, 100 and 50 μg/ml.Thus, the antioxidant activity of extract is similar to ascorbic acid activity at 200,100 and 50 μg/ml concentrations but no 25 μg/ml concentration, it is significantly decreased than ascorbic acid activity at this concentration (25 μg/ml), as recorded in Table 4.

Antidiabetic Activity
Twenty mice were divided into four groups (5 mice for each group) to study the antidiabetic activity of Marjoram extract.Group one represents the control group, the second group was injected with alloxan to convert to induced diabetes without treatment.Group three represents diabetic group with alloxan and treated with 0.5ml of DMSO containing 4 mg of extract daily for two weeks.Group four represents normal mice that injected with Marjoram extract.Table 5 shows the results of glucose levels in all the studied groups.The results revealed that glucose level was increased in diabetic group (G2) (174.982± 6.950).While the level of glucose in treated diabetics with marjoram extract dose (G3) is decreased toward normal case (108.172± 3.780).At the same time, the same dose of the extract was injected to the normal mice group (G4) but the glucose level was not affected by extract dose in comparison with control group.

Different small letters referred to significant differences between groups at p value less than 0.05
The results of insulin in all the studied groups are reported in Table 6.The results showed that insulin level was decreased in diabetic group G2 (14.Different small letters referred to significant differences between groups at p value less than 0.05 Table 7 shows the results of lipid profile of all the studied groups.The results of TGs revealed that the level of TGs in diabetic group G2 (138.230±6.65 a ) was increase in comparison with control group G1 (74.338 ±3.452 c ), whereas, TGs level in diabetic group with extract dose G3 was decreased toward normal level (97.552 ±2.625 b ).Also, TGs level in normal group with extract dose G4 (75.80 ±6.505 c ) does not affected by extract dose related to control group.The results of total cholesterol level (TC) showed significant increasing (109.629±6.58 a ) in diabetic group (G2) related to control group (G1) (66.138 ±3.452 c ).At the same time, TC level in treated diabetic group (G3) significantly decreased (76.482 ±2.850 b) in comparison with diabetic group (G2).In contrast, TC level in treated normal group (G4) was not affected with marjoram extract dose (68.252 ±2.721 c ), it was found to be similar to its level in control group (G1).
The results of HDL level showed significant decrease (18.367Different small letters referred to significant differences between groups at p value less than 0.05 The levels of AST and ALT were recorded in Table 8.The results showed that AST level increased in diabetic group G2 (153.370 ± 7.750 a ) compared to the control group G1(139.600 ± 13.10 b ).While the treated group with marjoram extract dose G3 decreased to normal level (145.420± 5.330 ab ).Also, the level of AST in normal group G4 that treated with extract group was not affected by Marjoram extract as shown in Table 4.In another hand, ALT level in diabetic group G2 (132.040 ± 13.30 a ) increased related to the control group G1(69.22 ± 4.650 c ), whereas, ALT level in the treated group with extract dose decreased toward normal case (112.260± 11.88 b ).It was noticed that the level of ALT in normal group does not affect by the extract dose and maintained its normal level as in the control group (66.70 ± 2.93c), as shown in Table .8. Different small letters referred to significant differences between groups at the same column at p value less than 0.05 Table 9 shows the testosterone levels in all the studied groups.The diabetic group G2 revealed decreasing in testosterone level (3.194 ± 0.188 c ) in comparison with control group G1(.11.652 ± 1.219 a ).In contrast, the level of testosterone in the treated group with extract dose G3 increased toward the normal state (8.369 ± 0.458 b ), while its level in normal group that treated with extract dose is not affected with extract (11.613 ± 0.670 a ), as shown in Table 9. Different small letters referred to significant differences between groups at the same column at p value less than 0.05

Discussion
The study of the antioxidant activities of plant compounds showed the important role of these compounds in the treatment of oxidants present in the biological system.Phenols represent the main compounds in plant extracts that possess antioxidant activity due to their structure containing aromatic rings that enable them to be stable and capable of donating hydrogen atoms to neutralize free radicals without being affected 35 .Accordingly, the qualitative tests of marjoram extract confirmed the presence of flavonoids and terpenoids which represented the major source of the antioxidant components.So, the results of qualitative tests of marjoram extract support the results of the antioxidant activity of the extract 34 .
Bacterial resistance to common antibiotics has led to a search for new antibiotics.Plants contain many and varied chemical compounds that have various properties and activities against infections, microbes and various diseases.The biologically active plant ingredients are safe and have a proven ability to treat various diseases.Furthermore, the effect of plant extracts on bacteria species differs due to the structure of bacterial cell membranes and the nature of the effective components of the extract.Generally, the mechanism of plant extract effect on bacteria includes the effect on the bacterial membrane to penetrate it, caused inhibit the DNA and protein synthesis, in addition to inhibiting a number of metabolic enzymes such as succinate dehydrogenase and malate dehydrogenase, increasing the osmotic pressure inside the cell, and organic acids can also lead to increase in the pH inside the cell.However, cell membrane damage is considered one of the main pathways affecting and inhibiting bacterial cells 36 .
The acute toxicity of plant extract should be determined by LD50 method.So, evaluating the LD50 value guides to determine the permissible doses to indicate their effect on the disease or ailments to be treated.LD50 value of marjoram extract is found to be 50.84mg/kg BW, which is considered a high value that can give permissible limits equal to 12.5 mg / kg body weight.However, the dose of 4 mg/ml was used as a value within the permissible limits for the treatment of diabetes and against pathogenic bacteria as upper doses with other lower doses include 2,1 and 0.5 mg/ml, which are all be under permissible dose limits 37 .
It was reported that plant extracts have high activity as antidiabetic factors.The activity includes glucose transport and metabolism regulation.Health advantages of plant extracts occurred due to presence of active bio components called the phytochemical compounds which are including flavonoids, terpenoids, alkaloids and phenolic acids like ellagic acid and ferulic acid.So, the presence of such compounds support the antidiabetic activity of the plant extract 38 .Furthermore, plant extracts lead to regulate blood glucose, in addition, the antioxidant and anti-inflammatory features suppressed lipid oxidation that can be associated with insulin resistance 39 .It was found that plant extract mechanisms of their actions to be as antihyperglycemic factors, occurred through utilized their properties in suppression of glucosidases, rise in glucose uptake and elevation of insulin secretion Studies referred to that plant extracts possess beneficial roles in the reduction of TC, LDL-C, and TGs levels and elevation of HDL-C levels, due to its function in reverse cholesterol transport in addition to its connection with antioxidant enzymes like paraoxonase 1that can reduce pro-atherogenic components that can may occur during lipid oxidation.Subsequently, marjoram extract regulates the lipid profile in the diabetic group in comparison with the control group and has no effect on the normal group that was treated with extract under the same condition 41 .Plant extract also found to be enhanced kidney and hepatic functions by decreasing the abnormal levels of GOT, GPT during diabetes period to the normal levels during the treatment period.Plant extracts activate SOD, GSPx and GSH, so they protect the cells from damage that occurred by free radicals.Moreover, it was found that certain plant extracts decreased the liver fibrosis in terms of decreasing collagen 1 and connective tissue 42 .
In diabetic rats, plant extracts like Alpinia officinarum extract improved sperm damage and its morphology in addition to repairing the histological defects in the testis 43 .In another study, methanolic extract of equisetum arvense treats the diabetic adverse effects on fertilization and sperm quality, so, this action may be linked with its bioactivities like antioxidant and anti-hyperglycemia activities 44 .
In recent study, it was reported that antidiabetic activity was increased with increasing of treatment time and the applied dose of plant extract 28 .This study is agreeing with current study in reducing the level of glucose and lipid levels.In another recent study, the activity of medicinal plant extracts attributed the active components represented by the secondary metabolites like flavonoids, phenolic acids, tannins, saponins and glycosides.So, the activity of this raw materials encourages to convert they into pharmaceutical agents 45 .Antidiabetic activity of the plant extract of Euphorbia neriifolia was investigated.The results showed increasing in beta cells numbers and insulin secretion.This result is consistent with the obtained results in current study 46.

Conclusion
On the basis of the obtained results in the antioxidant activity of this extract, it can be concluded that the methanolic leaves extract of marjoram has high antioxidant capacity compared to ascorbic acid.This activity attributed to the presence of phytochemicals in extract like polyphenol and terpenoids.Also, the results revealed the powerful antimicrobial activity of the marjoram leaves extract that it can be stopping or inhibit the growth of the studied species of pathogenic bacteria and fungi.So, using this extract as an antidiabetic agent support the immunological system of diabetic patients.Moreover, the study of antidiabetic activity showed that marjoram extract has the ability to reduce the level of blood sugar in diabetic mice and improve lipid profile, liver function and fertility disorders.Further studies should be conducted to isolate and characterize the pure active phytochemicals for improvement and supporting these bioactivities are recommended.

Figure 1 .
Figure 1.Shows the relation between mice death percentage and extract concentration for LD50 determination

Table 6 . Insulin level in all the studied groups
858 ± 1.482 b ). at the same time, glucose level was improved in diabetic group that treated https://doi.org/10.21123/bsj.2024.9328P-ISSN: 2078-8665 -E-ISSN: 2411-7986 Baghdad Science Journal with extract dose G3 (19.649 ± 1.966 a ) in comparison with control group G1 (21.177 ± 1.477 a ).also, the results showed that insulin level is not affected by extract in normal mice group that treated with extract dose G4 (21.802 ± 1.305 a ).