Activity of Moringa Oleifera Lam on Liver Function and Histology in White Male Rats

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INTRODUCTION
Moringa oleifera Lam is one of the herbs that can be used as food and medicine.Moringa oleifera Lam originated from the sub-Himalayan regions of India, Pakistan, Bangladesh, and Afghanistan and then spread to various areas, including Indonesia.Moringa oleifera Lam is often called the miracle tree because almost all plant parts, including the leaves, have extraordinary benefits.Moringa oleifera Lam leaves are commonly used to treat hyperlipidemia, cancer, arthritis, prostate problems, and rheumatism. 1 Moringa oleifera Lam leaves contain secondary metabolite compounds, such as alkaloids, flavonoids, phenolics, and triterpenoids/steroids.Alkaloids act as antimicrobials, and phenols as antioxidant compounds. 2Moringa oleifera Lam leaves contain quercetin, a flavonoid compound with antioxidant activity five times stronger than vitamin C and vitamin E. Antioxidants neutralise free radicals so that oxidative damage to biomolecules can be prevented. 3Other metabolites contained in Moringa oleifera Lam leaves include phenolic acids, carotenoids, polyphenols, isothiocyanates, phytates, glucosinolates, and phenolic acids. 4ringa oleifera Lam leaves have many pharmacological activities, such as antimicrobial, antioxidant, antihyperlipidemic, antiinflammatory, anticancer, antihyperglycemic, anti-hyperuricemia, and analgesic. 5Dillasamola et al. 6 reported that Moringa oleifera Lam leaves have immunomodulatory activity as immunostimulants ethanol extract of Moringa leaves at 10, 30, and 100 mg/kg BW doses.Immunomodulators are substances that alter the function of the immune system by dynamically controlling immune cells, such as cytokines.Immunomodulators work by restoring impaired immune function (immune restoration), improving immune system function (immunostimulation), and suppressing immune responses (immunosuppression).There are two types of immune responses when exposed to foreign substances: specific and non-specific.The specific immune response is the immune response that happens after exposure to specific foreign substances.In contrast, the non-specific immune response is the innate immune response that has existed since birth. 7man et al. 8 reported that the LD50 of Moringa oleifera Lam leaves ethanol extract in rats was 6616.67 mg/kg BW.The traditional use of Moringa oleifera Lam leaves in the health sector, significantly as an immunostimulant, is increasing among the public to overcome various diseases.However, scientific research on the safety of long-term and regular use of Moringa oleifera Lam leaves is still limited.Thus, researchers are interested in conducting a subacute toxicity test of Moringa leaves for 21 days and observing liver histology and ALT enzyme activity in male white rats.The tested Moringa oleifera Lam leaves were extracted using standardised ethanol solvent.The results of this study are expected to be an overview of the safety of Moringa oleifera Lam in repeated use and routine use and can be used as a basis for further research.

Preparation of extraction
5 kg of fresh Moringa oleifera Lam leaves samples were sorted and cleaned from impurities, then air-dried to obtain dry samples.Afterwards, powder making was carried out with a grinder until a fine powder of Moringa oleifera Lam leaves was obtained. 9The powder was macerated using 70% ethanol solvent (1:10) for 24 hours with three times repetitions.The powder was soaked for the first 6 hours while occasionally stirred and left undisturbed for 18 hours.All the macerates were collected and evaporated using a rotary evaporator to obtain a concentrated extract.

Organoleptic test
The extracts obtained were tested organoleptically using sensory that assessed the extract's form, colour, taste, and odour. 9

Yield
Calculation of yield was performed by weighing the Moringa leaf crude drug (simplified) to obtain mass (A), then weighing the obtained extract to obtain mass (B). 9 The yield was calculated using the formula: Determination of total ash content Put 2-3 g of the extract in a crucible that has been incinerated and tared, gently incandescent until the charcoal is exhausted, cool and weighed.If, in this way, the charcoal cannot be removed, add hot water, stir, and filter through filter paper along with the filtering residue in the same crucible.Put the filtrate into the krus, evaporate and incandesce until the weight remains at 800±25 °C.Calculate the ash content of the material dried in the air with the following equation. 9

Determination of acid-insoluble ash content
Boil the ash obtained to determine total ash content with 25 mL of dilute hydrochloric acid LP for 5 minutes.Collect the acid-insoluble part, filter through ash-free filter paper, wash with hot water, and incandesce in a crucible until the weight remains at 800±25 °C.Calculate the acid-insoluble ash content of the air-dried material with the following equation. 9termination of moisture content (gravimetric method) Weigh approximately 10 g of extract in a pre-weighed container.Dry at 105 °C for 5 hours and weigh.Continue drying and weighing at onehour intervals until the difference between two consecutive weighings is less than 0.25%. 9ytochemical screening of Moringa oleifera Lam extract Flavonoid examination Put 1 mL of extract in a test tube, then add a few drops of concentrated HCl and a little Magnesium powder.The orange-red to purple-red indicates that the extract contains flavonoids. 10

Phenolic examination
Put 1 mL of extract into a test tube, then add FeCl 3 1%.The formation of a blue-black colour characterises positive results. 2

Saponin examination
Put 1 mL of test extract into the tube, and shake vertically for 10 seconds.If a stable foam forms, it is positive for saponins. 11

Steroid and triterpenoid screening
Dissolve the extract in 3 mL of chloroform, then add 2 mL of anhydrous acetic acid and 2 mL of concentrated sulfuric acid through the tube wall.If a brownish or violet ring is formed at the restriction of two solvents, it indicates the presence of triterpenoids, while if a bluishgreen colour is formed, it indicates the presence of steroids. 11

Alkaloid screening
Take a few mg of extract and add a few drops of Wagner's reagent.Positive results are characterised by forming a brown precipitate. 2

Dose planning
The ethanol extract of Moringa oleifera Lam will be administered to male white rats at 7, 21, and 140 mg/kg BW.The dose was followed by the immunostimulant activity test of Moringa oleifera Lam leaf ethanol extract, which uses 10, 30, and 100 mg/ kg BW in male white mice.(Dillasamola et al., 2018)..There is a difference in the highest dose used because the researchers want to observe the activity when the dose was increased to 140 mg/kg BW and whether it caused any toxic effects on the liver.The test formulations will be administered orally daily for seven days, 14 days, and 21 days.

Preparation of 1% Na CMC suspension
Weigh 500 mg of Na CMC and sprinkle it on 10 mL of hot water in a heated mortar.Leave it for 15 minutes and then grind until homogeneous.Next, add aquadest until the volume is 50 mL.

Preparation of suspension of Moringa leaf ethanol extract (Moringa oleifera L.)
Suspend 1 g of extract into 50 mL of 1% Na CMC.Then, dilute the test suspension according to the concentrations of each designed dose.The Afniting, the glass slide is rubbed with Mayer's albumin.Place several incisions on it, drip with water, and stretch on a hot plate until the incisions expand.Staining with hematoxylin Erlich and eosin was performed as follows: Xylol I, xylol II (5 minutes each), absolute alcohol I, absolute II, 80%, 95%, and 100% (3 minutes each), Ehrlich's hematoxylin for 10 minutes, then washed with running water.Next, the preparations were dipped in eosin for 5 minutes, absolute alcohol III and absolute IV (each for 2 minutes), xylol III and xylol IV for 1 minute, and washed with running water.Mounting (closure) so that the tissue does not dry, eating is dripped with adhesive in the form of balsam Canada, covered with cover glass, and then dried.And then, label the preparation to the right of the object, examine it under a microscope and take a microscopic photograph. 13

Examination of liver histology preparations
Examination of liver histology preparations with 400x magnification.The assessment of histological damage is semi-quantitative and is based on scoring (values) according to the observed changes in the liver organ under a microscope, then scoring based on the matrix in Table 1. 14

Data analysis
The data were analysed using a two-way ANOVA statistical test between time (duration of administration) and dosage.Furthermore, the data were analysed with Duncan's Multiple Range Test).This process was conducted with IBM SPSS.

Extraction process
The extraction process was performed using the maceration method.The maceration method was chosen because it allows for a large sample size with a simple procedure, requiring no special treatment, and is suitable for heat-sensitive compounds. 15This maceration process uses a brown bottle placed in a place protected from light.This process aimed to avoid the decomposition of the active substance structure.Moringa oleifera Lam leaf simplisia powder was macerated using 70% ethanol as the solvent.Ethanol was chosen because it is a relatively safe universal solvent that can dissolve almost all polar, semi-polar, and non-polar compounds.70% ethanol was selected because the study utilised dried samples with relatively low water content.The 30% water content of this solvent serves to help break the cell wall so that ethanol penetration into the cell is faster and more optimal. 16e maceration process was carried out for 24 hours with three repetitions with a ratio of sample and solvent of 1:10.The powder volume of the test formulation injection should be adjusted based on the body weight of the test animals.

Administration of test preparations
The animals will be divided into four groups as follows: Group I: Only given 1% Na CMC Group II: Given the extract at a dose of 7 mg/kg BW Group III: Given the extract at a dose of 21 mg/kg BW Group IV: Given the extract at a dose of 140 mg/kg BW Each group consists of 9 rats, further divided into three subgroups: subgroup A (treated for seven days), subgroup B (treated for 14 days), and subgroup C (treated for 21 days).Each subgroup consists of 3 rats.
On days 8, 15, and 22, the animals are anesthetised and partially conscious, and blood samples are taken through the orbital sinus of the eye to determine the ALT activity.Then the animals were sacrificed by inhalation anaesthesia using ether.They were then dissected, and their liver was taken to prepare histological slides using the paraffin method.These slides were observed under a microscope.

Blood serum collection
Thirty-six test animals were sacrificed and collected the blood on days 8, 15, and 22 after the experiment.The blood was collected through the orbital sinus of the eye.Then, the blood was collected in a gel activator tube and centrifuged for 10 minutes at 3000 rpm to obtain serum.The clear solution of serum was then pipetted using a micropipette and transferred into microtubes.A 5010 v5+ photometer immediately read the serum to check the ALT levels of the test animals.

Monoreagent preparation
Mix four parts of reagent 1 with 1 part of reagent 2 (20 mL of reagent 1 + 5 mL of reagent 2) = mono reagent. 12T level examination 100 µL serum and 1000 µL mono reagent were put into a test tube to be homogenised for 1 minute.Measure the blank's absorbance value before checking the serum's ALT levels.Then, the serum and mono reagent mixture was immediately measured for absorbance using a 5010 v5+ photometer right after the 1st minute at a wavelength of 340 nm.Record the results shown on the photometer. 12

Histology preparation
To begin with, sacrifice the rats, remove the liver, and rinse with physiological NaCl solution.Transfer the organ into a formalin buffer solution.Then, cut the liver organs into several sections representing the entire liver.Organ tissues were dehydrated with 80%, 95%, and absolute alcohol solutions for 1 hour each.In addition, transfer the object into a solution of absolute alcohol: xylol (1:1), xylol 1 and xylol 2, each for 1 hour for clarification.Furthermore, place the object into the infiltration solution in an incubator at 56-600C.Xylol: paraffin, paraffin I, paraffin II, and paraffin III (each for half an hour).Next, embedding, the object is inserted into a metal mould or paper box filled with liquid paraffin that was heated in an incubator and then allowed to cool and freeze.Section by placing a paraffin block in a holder, then thinly slice with a microtome knife (5-6 um).was soaked for the first 6 hours while occasionally stirred and left undisturbed for 18 hours.All the macerates were collected and evaporated using a rotary evaporator to obtain a concentrated extract weighing 116.1 grams.

Organoleptic test of purified extract
The organoleptic examination of the ethanol extract of Moringa oleifera Lam leaves revealed a concentrated extract with a dark greenish-brown colour, distinct aroma, and a bitter taste.These results comply with the organoleptic standards for Moringa oleifera leaf extract in the Indonesian Herbal Pharmacopoeia. 9

Yield of purified extract
Furthermore, calculating the percentage yield of Moringa oleifera Lam leaf ethanol extract yielded 17.86%.The yield was calculated to determine the percentage of extract obtained from the initial sample weight and determine the solvent's ability to attract active substances in the sample.The percentage value of the yield obtained has met the standard yield percentage in the Indonesian Herbal Pharmacopoeia, which is not less than 9.2%. 9

Preliminary examination of chemical content (phytochemical screening)
The ethanol extract of Moringa oleifera leaves is a method used to determine the metabolic content in a plant.Qualitative phytochemical screening of the ethanol extract of Moringa leaves indicated the presence of phenolic compounds, flavonoids, alkaloids, and steroids/ triterpenoids.Demographic conditions, nutrition and the environment where a plant grows are factors that cause differences in the content of chemical compounds.

Examination of moisture content, total ash content and acid-insoluble ash content
The examination of moisture content in the ethanol extract of Moringa oleifera leaves resulted in 9.15%.This moisture content value meets the requirements specified in the Indonesian Herbal Pharmacopoeia, which should not exceed 10%. 9The examination of total ash content yielded 5.01%.The ash content value meets the requirements in the Indonesian Herbal Pharmacopoeia, which is no more than 9%. 9Meanwhile, the examination of acid-insoluble ash content yielded 0.469%.This acidinsoluble ash content value complies with the requirements in the Indonesian Herbal Pharmacopoeia, which should not exceed 0.9%. 9

Administration of test preparations
Each group consisted of 9 rats divided into three subgroups: Group A, which was treated for seven days; Group B, which was treated for 14 days; and Group C, which was treated for 21 days.Each subgroup consisted of 3 rats.Rats in Group 1 were designated as the control group and were given a 1% Na CMC suspension.The 1% Na CMC suspension was used as a suspending agent because it is inert, nontoxic, non-irritating, and produces a stable solution.Groups II, III, and IV were administered test preparations at 7 mg/kg BW, 21 mg/kg BW, and 140 mg/kg BW, respectively.
On days 8, 15, and 22, the animals were half-conscious, and their blood samples were taken through the orbital sinus of the eye to determine ALT levels.Subsequently, the animals were euthanised by inhalation anaesthesia using ether, then dissected, and the liver was taken to make histological preparations using the paraffin method and observed under a microscope.

Analysis of ALT activity
ALT enzyme is a specific indicator of liver damage.ALT enzyme is produced in the liver and released into the blood, where its level is directly proportional to the condition of the liver.Higher levels in the blood indicate more severe liver damage. 17The examination of ALT levels was carried out using a 5010 v5+ photometer with an enzymatic method.The principle of determining ALT kinetics was based on IFCC (International Federation of Clinical Chemistry) recommendations, where a chemical reaction occurs between L-alanine contained in reagent 1 with 2-oxoglutarate contained in reagent 2. The ALT enzyme catalysed the chemical reaction in the experimental animals' serum.This chemical reaction produces L-glutamate and pyruvate.Then the pyruvate formed will be reduced by NADH contained in reagent 2 with the help of the LDH catalyst in reagent 1 to produce D-lactate (Greiner Diagnostic GmbH). 18According to BPOM, the standard value of ALT levels in rats is 10-50 IU/L. 19sed on the statistical analysis of ALT levels using two-way ANOVA, it was found that the average ALT level was significantly influenced by the dose and duration of Moringa oleifera Lam leaf extract administration (p<0.05).However, the interaction between the dose and duration of Moringa oleifera Lam leaf extract administration did not significantly affect the average ALT level (p>0.05).
Furthermore, the DMRT result showed a non-significant difference in mean ALT levels in the 7 mg/kg BW dose group against the control group.However, in the dose group of 21 and 140 mg /kg BW, there is a significant difference in average ALT levels compared to the control group, with an increase in ALT levels beyond the normal range.
Duncan's test on the duration of Moringa oleifera leaf extract administration showed a significant difference in the average ALT level between the group treated with the extract for seven days and the groups treated with the extract for 14 days and 21 days.The groups treated with the extract for 14 days and 21 days did not show a significant difference in the average ALT level, but there was an increase in the ALT level above the normal range.
Based on the obtained range of results, the average ± SD ALT levels for the control group and the groups administered Moringa oleifera Lam leaf extract at doses of  2).Based on Table 2, the ALT levels slightly increased above the normal range.It happened due to lipophilic compounds in Moringa oleifera Lam leaf extract, such as alkaloids and flavonoids, which can damage the liver by disrupting the liver cell membrane and increasing membrane permeability, increasing ALT levels. 20

Liver ratio value calculation
Calculating the liver organ ratio values is used as a supporting parameter.It was because changes in organ weight are sensitive indicators of organ changes caused by chemicals.A compound can affect both an animal's organs and overall body weight.Therefore, a ratio value called relative organ weight was calculated by dividing the weight of each animal by its body weight. 21ts sacrificed by inhalation anaesthesia using ether were dissected to take the liver and then weighed to calculate the liver organ ratio.The liver organ ratio observations showed no significant effect of dose and dose interaction with duration of administration (p>0.05).However, the duration of Moringa oleifera Lam leaf ethanol extract had a significant effect on the ratio of liver organs (p<0.05).
Furthermore, DMRT results on the duration of ethanol extract administration from Moringa oleifera Lam leaves showed a significant difference in the average value of organ ratios in the test group given moringa leaf ethanol extract for seven days, 14 days and 21 days.The 21-day administration of the ethanol extract from Moringa oleifera Lam Afriwardi A, et al.Activity of Moringa Oleifera Lam on Liver Function and Histology in White Male Rats leaves significantly decreased the average liver-organ ratio, followed by the 14-day and 7-day administrations.
Administration of Moringa oleifera Lam leaf extract dosage in the treatment group at 140 mg/kg BW decreased the liver-organ ratio compared to the control group.The average ± SD values of the liver organ ratio in the control group, dosage of 7 mg/kg BW, 21 mg/kg BW, and 140 mg/kg BW were 0.038 ± 0.009, 0.040 ± 0.008, 0.039 ± 0.010, and 0.038 ± 0.008, respectively.Meanwhile, the average ± SD values of the liver organ ratio for 7 days, 14 days, and 21 days were 0.046 ± 0.006, 0.039 ± 0.005, and 0.031 ± 0.006, respectively (Table 3).An anomaly in the control group parameter was the difference in liver organ ratio values.It was due to the experimental animals having varying body weights.Differences in organ weight are often accompanied by differences in body weight between treatment groups, making interpreting organ weight more difficult.Therefore, the liverorgan ratio is only used as a supporting parameter that helps indicate cell damage.

Liver histology analysis
Liver tissue preparations were observed using a light microscope at a magnification 400x.surrounding lobules, a score of 5 was given. 14The examination results were described descriptively by comparing the histological damage in the liver between the treatment group and the control group.
Histology of the liver of the test animals for 7 days showed that the control group and the dose of 7 mg/kg BW showed liver tissue with parenchyma containing hepatocytes in lobules arranged regularly within normal limits so that it was given a score of 0 (Figure 3. Histology of the liver of the test animals for 14 days showed that the control group and the dose of 7 mg/kg BW showed liver tissue with parenchyma containing hepatocytes in lobules arranged regularly within normal limits so that it was given a score of 0 (Figure 4.  0,046 ± 0,008 0,040 ± 0,005 0,029 ± 0,004 0,038 ± 0,009 7 mg/kg BW 0,046 ± 0,006 0,038 ± 0,005 0,035 ± 0,008 0,040 ± 0,008 21 mg/kg BW 0,047 ± 0,007 0,039 ± 0,008 0,031 ± 0,009 0,039 ± 0,010 140 mg/kg BW 0,046 ± 0,004 0,037 ± 0,004 0,030 ± 0,006 0,038 ± 0,008 Mean ± SD 0,046 ± 0,006 0,039 ± 0,005 0,031 ± 0,006 Table 3: The effect of dosage and duration of Moringa oleifera Lam leaf extract administration on the liver organ ratio values in male white rats.The observed liver tissue preparations were taken from the area near the central vein.The central vein allows more evident observation of damage due to its larger size than other blood vessels.Additionally, the central vein is where blood enters the liver tissue. 16Cells close to the central vein contain a high concentration of metabolites due to the area around the central vein being more often damaged than the peripheral area.In this study, the shape of the central vein was observed to widen with increasing dosage and duration of administration.However, at the 21-day duration, the central vein appeared to shrink.It is due to stasis in the sinusoids, which begins to experience oedema.The table below presents the liver histology scores from 5 fields of view (FOV).
Histological assessment of the toxicity effect of Moringa oleifera Lam leaf ethanol extract on liver histology showed histological differences between the control group and the treated group.In the negative control group, the parenchyma consisted of hepatocyte cells arranged regularly in trabeculae and separated by sinusoids, with no signs of degeneration or inflammation.In the low and medium-dose treatment groups, 7 mg/kg BW and 21 mg/kg BW showed a picture of liver tissue within normal limits to minimal damage (Table 4).It indicates that Moringa oleifera Lam leaf extract is relatively safe at doses 1 and 2. However, administering a high dose for 14 and 21 days showed histological changes in the liver, although only minimal to mild damage was observed (Table 4).It suggests potential adverse effects of Moringa oleifera Lam leaf extract at high doses and long term on liver tissue.Throughout the 21-day study period, no toxic symptoms were observed in the test animals, and no sudden deaths were recorded.

CONCLUSIONS
Based on the description above, it concluded that administering ethanol extract of Moringa oleifera Lam to male white rats increased   average ALT levels at a dose of 140 mg/kg BW for 21 days but still within normal limits.Furthermore, administering ethanol extract of Moringa oleifera Lam at 7 and 21 mg/kg BW doses for 7, 14, and 21 days showed regular liver tissue observations within the normal range.
The 400x magnification was used to observe changes in cell morphology.Liver histology damage scores were assessed globally on tissue preparations by evaluating the degree of hepatocyte necrosis.If no hepatocyte necrosis was observed in normal cells, a score of 0 was assigned.If the cells had minimal-mild damage, focal, limited to the centrilobular region, with <1/4 of affected lobules being necrotic, the score was 1.If the cells had mild-moderate damage, focal-multifocal, central-mid zonal lobular region, with ½ affected lobules necrotic, the score was 2. If the cells have moderate to severe damage, multifocal, centrilobular-portal region with more than ½ of the lobule and less than ¾ of the lobule affected by necrosis, a score of 3 was given.If the cells have severe damage, multifocal, with more than ¾ of the lobule affected by necrosis, a score of 4 was assigned.If cells showed severe damage (across the entire lobule), hepatocyte loss in the central vein area extending to the portal area and spreading to (a) and (b)).However, at doses of 21 mg/kg BW and 140 mg/kg BW, minimal focal damage was observed in the centrilobular area, with less than ¼ of the lobule showing necrosis, resulting in a score of 1 (Figure 3. (c) and (d)).

Figure 1 :
Figure 1: Graph of the effect of dose and duration of Moringa oleifera Lam leaf extract administration on average ALT activity in male white rats.

Figure 2 :
Figure 2: Graph of the effect of dosage and duration of Moringa oleifera Lam leaf extract administration on the liver organ ratio values in male white rats.