INTRODUCTION:

The Maternal Mortality Rate (MMR) in Indonesia was 305 per 100,000 live births in 2015 and it was considered high ¹^,². Factor that affecting maternal mortality is postpartum haemorrhage due to anaemia³. It has adverse effect such as uterine subinvolution, long term perineal wounds, pyrexia, puerperal infection, reduced breast milk production, stress, sepsis and maternal mortality⁴^,⁵. To prevent these conditions, post-partum mothers should fulfill nutritional needs by consuming iron rich foods^6-8.

There are natural plantsin Indonesia, especially in South Kalimantan can be used to treat anaemia, easy to obtain, process, low cost, and has benefit for health^9-12. One of the resources plants to treat anemia is Moringa oleifera leaves (Moringa oleifera Lam)¹³^,¹⁴. This leaveconstitutes as a supplement due to its richness in vitamins, such as beta carotene (Vitamin A), Vitamin B (pyridoxine, nicotinic acid, and folic acid,), Vitamin D, potassium (K), Vitamin E, Vitamin C, and mineral salts such as magnesium (Mg), copper (Cu), iron (Fe), zinc (Zn), calcium (Ca). In addition, this leaves also source of protein and lipids. The phytochemicals qualitative showed that this leaves also richness of carotenoids, flavonoids, phenolic compounds, tocopherols, ascorbic acid, folate, polyunsaturated fatty acids, amino acids, alkaloids, tannins, saponins¹⁵^,¹⁶.

If we consume moringa leaves directly, it hasbitter taste and it requires large amounts for each consumption¹⁷. Alternatives to consume this moringa leaves using Moringa leaf juice that was formulated into extract powder and putting it in capsules¹⁸^,¹⁹. However, it is necessary to optimize the bioactive compound of Moringa leaf extract capsules and investigate their efficacy in postpartum women.

MATERIALS AND METHODS:

Research design:

This research is an experimental study including phytochemical screening, determination active ingredient, capsule preparation, toxicity tests, and clinical trials of Moringa leaf extract capsules on among postpartum women.

Instruments:

The materials used are Moringa oleifera Lam leaves, 96% ethanol, gelatine, pro-analytical ethanol, FeCl3, lead acetate, hydrochloric acid, Mayer’r reagent, dragendorf, sterile aqua, Leiberman-Burchard, gallic acid, Folin-Cioceltau reagent (FCR), NaOH, starch, nipagin, nipasol, magnesium stearate, capsules, methanol, shrimp larvae and shrimp larvae media. Toolswere used were glassware, macerated vessel, waterbath, oven, laminar air flow, apparatus for evaluating toxicity, rotary, shrimp larvae vessel, hardness tester, Hb and HCT tool diagnosis

Extraction Process:

Fresh Moringa leaves were washed, weighed, and dried at 60°C for 72 hours. The powder was mixed with pro-analytical ethanol solvent with a ratio of 1:10 for 1x24 hours and we changed the solvent twice. The liquid extract obtained was filtered, then evaporated using a rotary evaporator and dried over a water bath.

Phytochemical Screening:

Phytochemical testing was carried out using extracts with a concentration of 1% or 1gram in 100ml.

Determinant active ingredients:

a) Flavonoid Levels:

The total flavonoid content of the ethanol extract, then adding the ethanol solvent p.a. in 10mL volumetric flask. Take 0.5mL of the ethanol extract solution and put it in a test tube with 0.5mL of 10% AlCl3 added and 4 mL of 5% acetic acid, then shake the solution and let it sit for the operating time. The absorbance obtained was read with a UV-Vis spectrophotometer instrument using the maximum wavelength²⁰.

b) Phenolic Content:

Gallic acid was used as a standard for making grade series. The standard gallic acid solution was prepared in series with concentrations of 20, 30, 40, 50, 60 and 70 ppm. Each concentration of gallic acid standard solution was taken as 0.5mL and put in a test tube then added with 2.5mL of 5% Folin Ciocalteau reagent, shaken until homogeneous and left for 8 minutes. Each solution was added as much as 2mL of NaOH solution, vortexed until homogeneous and let the rest until 30 minutes. All solutions were measured for their absorbance with a UV-Vis spectrophotometer at a wavelength of 742nm, then a standard curve was made using the equation y= bx + a, it means the relationship between gallic acid concentration (μg/mL) and absorbance. all the reagents were used with ethanol solvent. Samples are required for standard compounds²¹^,²².

Capsule preparation:

Capsule preparation was made from extract solution. The extract solution is mixed with starch as an additional ingredient, magnesium stearate as a lubricant, nipagin and nipasol aspreservative. Then it was baked in the oven until dry, grind it with a blender until obtain powder. The powder is put into capsules with a special tool.

Cytotoxicity Evaluation:

a) Research location for collecting of Artemia salina leach.

Collection of shrimp eggs was conducted in the Laboratory of the Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Lambung Mangkurat.

b) Preparation larvae of Artemia salina leach

Two days prior to the test, Artemia salina eggs were born. In order to hatch Artemia salina eggs, we created vessels. In order to hatch the eggs, 2.5mg of them were placed in a container with 250mL of seawater and a 25 watt bulb with an aerator. After 24hours, the eggs hatched and turned into larvae. A suitable leach for the Brine Shrimp Lethality Test (BSLT) test is ready to be used as a toxicity test target when it is less than 48 hours old. Pipetting into a beaker filled with seawater allowed us to separate the Artemia salina larvae from the eggs.

c) Concentration preparation of test samples

The test solutions for BSLT had concentrations of 1500, 1000, 500, 100, and 0g/mL (as controls). From tofu, we created a stock solution. The tofu was pulverized into an extract that weighed up to 1000 mg and was then mixed in 100 mL of seawater to create a stock solution with a 10,000 g/mL concentration. In addition, we made stock concentrations of 1500, 1000, 500, 100, and 0µg/mL.

d) Determination of LC₅₀

The natural ingredient must be tested using toxicity test. Toxicity tests can be carried out using the BSLT (Brine Shrimp Lethality Test) method. In this test, shrimp larvae were used as living model creatures. This test was simple, rapid, bench-top and describes the safety level of a material²³^,²⁴. Testing the activity of a natural substance must be accompanied by a toxicity test. Consequently, toxicity tests were run on each sample extract. Then, we made containers for each concentration of the sample extract. 4 containers were needed for the test and 1 container for the control. Then, at each concentration of the fluid, 10 Artemia salina larvae were added. Larvae observations were made for 24 hours and compared to the control group. The larvae have to remain motionless while being observed in order to be considered dead. The amount of toxicity was assessed after 24 hours of observation by counting the dead larvae. Linear Regression Analysis was used to calculate the LC50 value²⁵^,²⁶.

Clinical study of Moringa leaf extract capsules:

The next step was tested Moringa leaf extract capsules on postpartum women. All of procedure was granted by ethical clearance number 411/UMB/KE/VI/2023 from Universitas Muhammadiyah Banjarmasin Indonesia. The sample in this study was post-partum mothers who agreed join in this study and they must sign informed consent if agreed to join. We used snowball sampling to recruit the respondents. The sample were divided into two groups randomly. The haemoglobin and haematocrit levels were determined before treatment (pretest). Furthermore, the first group was given iron supplements every day for 10 days, while the second group was given Moringa leaf extract capsules every day for 10 days. On the 11^th day, haemoglobin and haematocrit levels were evaluated in posttest.

RESULTS AND DISCUSSION:

Indonesian have been consuming Moringa oleifera leaves for traditional medicine and food due to sources of nutrition and good for health. This plant contains nutrients such as copper (Cu), zinc (Zn), magnesium (Mg), iron (Fe), calcium (Ca), potassium (K), Vitamin A (beta carotene), Vitamin B (folic acid, pyridoxine, and nicotinic acid), Vitamin C, Vitamin D, and Vitamin E, protein and lipids. Phytochemical of Moringa oleifera leaves showed carotenoids, flavonoids, phenolic compounds, tocopherols, ascorbic acid, folate, polyunsaturated fatty acids, amino acids, alkaloids, tannins, and saponins²⁷^,²⁸.

Moringa oleifera leaves can be used as an alternative treatment for anemia. It can be consumed fresh, cooked or transformed into powder form. Previous studies showed that that Moringa leaf extract is a good source of antioxidant²⁹^,³⁰. Consuming Moringa leaf was safety evaluation on in vitro models and in vivo models³¹^,³². According to some research, moringa leaves have 25 times more iron than spinach and have seven times more vitamin C than oranges. They also have ten times more vitamin A than carrots, seventeen times more calcium than milk, and fifteen times more potassium than bananas³³. Another study showed that giving Moringa leaf capsules (500 mg/day) for 30 days increased hemoglobin (Hb) levels in postpartum mothers³⁴. The minimum dose is one capsule was 500 mg and the highest dose was four capsules (2000 mg) of extract or powder³⁵.

Phytochemical analysis of Moringa oleifera:

The results of qualitative phytochemical screening of Moringa oleifera are showed in table 1. Qualitative phytochemical screening is needed to measure bioactive compounds. The metabolites analysis includes flavonoids, phenolics, alkaloids, tannins, saponins, steroid, terpenoids. This study in line with previous studies which mentioned that Moringa oleifera leaves contain bioactive compound^36-39. These active compounds have benefit in human.

Table 1: Qualitative Phytochemical screening of M. Oleifera

Phytochemical	Reagent	Result
Flavonoids	Magnesium andAcid chloride	+
Phenolics	Ferric Chloride	+
Alkaloids	Reage Meyer	+
Tannins	Hydrolyzed collagen	+
Saponins	Aquades	+
Steroid	Liebermann Burchard	+
Terpenoids	Liebermann Burchard	+

Active ingredients of Moringa oleifera:

The extract was measured for secondary metabolite levels with phenolic and flavonoid parameters. Active ingredients of are presented in Table 2.

Table 2: Active Ingredients of Moringa oleifera Extract

No.	Active compound	Standard	Replication	Concentration	Percentage of concentration
1.	Phenolic	Galat acid	1	132.13 mg/g	13.21%
			2	136.41 mg/g	13.64%
			3	135.59 mg/g	13.55%
Mean				134.71 mg/g	13.47%
2.	Flavonoid	Quercetin	1	43.78 mg/g	4.37%
			2	40.63 mg/g	4.63%
			3	40.86 mg/g	4.08%
Mean				34.11 mg/g	4.17%

Table 2 showed that the phenolic of Moringa oleifera leaf extract was 13.47%. It means every 100 parts of the extract, there were 13.47 parts of phenolics. These results showed high total phenolics in the extract. These study is higher than previous study which showed phenolic was 12.78% and 9.94%⁴⁰. Further, the flavonoid of Moringa oleifera leaf extract was 4.17%. This result showed that 100 parts of the extract , there were 4.17 parts of flavonoid. It was higher than previous study: 3.56%⁴¹.

Formulation of extract capsule:

The extract is formulated with several compound in capsules. The capsule preparation formula was presented in table 3.

Table 3: Moringa Leaf Extract Capsule Formula

Ingredients	Function	Weight
Moringa Leaf Extract	Active compound	50 mg
Strach	Filler	233 mg
Magnesium stearate	Lubricant	15 mg
Nipagin	Preservative	1 mg
Nipasol	Preservative	1 mg
	Total	300 mg

The formula was obtained from review study then we tested to optimize the formula. The capsules are made in herbal medicine factory that has been certified by Indonesian Food and Drug Authority using industry-scale manufacturing techniques. The ingredients used are made in a capacity of 1000 capsules. The results of this formula were capsules containing Moringa oleifera extract which can be consumed directly.

CytotoxicityEvaluation:

Tabel IV: Brine shrimp lethality test (BSLT)

Concentration (µg/ml)	% mortality
Concentration (µg/ml)	1^st Replication	2^nd Replication	3^rd Replication
100	10	0	0
500	10	20	20
1000	20	30	20
1500	40	40	30
LC₅₀	2159,37 µg/ml	1781,81 µg/ml	2150,27 µg/ml
Mean LC₅₀	2150.27 µg/ml
Result (> 1000 µg/ml)	Not Toxic

The aim of cytotoxicity evaluation was to determine possible toxicity that may occur in the preparation. Testing was carried out using three replications to ensure validity and reliability test. Tests were carried out at 4 graded concentrations to determine the possibility of death of shrimp larvae at various concentrations. The results showed that the average LC₅₀ value was 2150.27µg/ml. The literature showed that, LC50 > 1000 µg/ml means not toxic⁴². Based on the result of toxicity tests capsules of Moringa oleifera leaf extract can be used on human model.

Clinical study of Moringa oleifera leaf extract capsule for post-partum mothers:

Moringa oleifera leaf extract capsule was evaluated on a limited number of human subjects. The samples were postpartum mothers. We had two groups, experiment and control. The experiment obtained the Moringa oleifera leaf extract capsule. Meanwhile the control group was obtained iron supplement. Hemoglobin and hematocrit levels were determined before the intervention (pretest). The treatment was conducted for 10 days, after treatment the hemoglobin and hematocrit levels were measured again (post-test). The results of hemoglobin and hematocrit are presented in table 5 and table 6.

Table 5: Distribution of Hemoglobin on both groups

Respondent	Control		Experiment
Respondent	Pre	Post	Pre	Post
1	8.4	9.8	10.8	13.3
2	10.5	13.6	8.1	13.2
3	12.9	12.2	7.8	11.5
4	13.6	12.2	10.8	12.2
5	13.2	14.2	11.5	14.2
6	11.5	11.2	11.3	13.6
7	10.5	9.1	11.2	12.9
8	14.9	13.6	9.8	12.5
9	11.5	14.6	7.1	10.5
Mean	11.88	12.27	9.82	12.65
Percentage	3.27%		28.84%

Tabel 6: Distribution of Hematocrit on both groups

Respondent	Control		Experiment
Respondent	Pre	Post	Pre	Post
1	25	29	32	40
2	31	40	24	39
3	38	36	23	34
4	40	36	32	36
5	39	42	34	42
6	34	33	33	40
7	31	27	33	38
8	44	40	29	37
9	34	43	34	31
Mean	35.11	36.22	30.44	37.44
Percentage	3.16%		22.99%

Table 7: Mean difference parameters

Parameters	Control		Experiment		Sig
Parameters	Baseline	Post intervention	Baseline	Post intervention	Sig
Hemoglobin	11.88	12.27	9.82	12.65	0.071
Hematocrit	35.11	36.22	30.44	37.44	0.075

The result showed that the hemoglobin levels of both groups before treatment were lower than after treatment. There was an increase in the average hemoglobin level before and after treatment. It means increased in hemoglobin levels when giving iron supplements or Moringa oleifera leaf extract. The increase of hemoglobin levels in control group was 3.27%, while in experiment group was 28.84%. It showed the ability of Moringa leaf extract capsules was effective to increase hemoglobin levels. This study in line with previous study which mentioned that Moringa oleifera leaf can increase hemoglobin levels in postpartum mothers³⁴. The reason was Moringa oleifera leaf contain high vitamin, mineral, and protein³⁴.

The statistical test of hemoglobin and hematocrit were presented in table 7. Using the Shapiro-Wilk and Kolmogorov-Smirnov procedures, do a normality test. (Sig > 0.05) It demonstrated that the data were regularly distributed. Levene Test homogeneity analysis. The results were likewise homogenous (Sig > 0.05). So, we used One Way Anova. Based on statistical tests, it is known that the hemoglobin levels in the group before giving iron supplements and Moringa leaf extract capsules were not significant. The results of hemoglobin levels after obtained iron supplement and Moringa oleifera leaf extract capsules were also not significant; however, the mean level of hemoglobin was increase (Table 5). It means that Moringa leaf extract capsules can be an alternative among postpartum mothers with anemia. This is in accordance with previous research that Moringa leaf capsules can increase hemoglobin levels in postpartum mothers³⁴. Data on hematocrit showed there was no significant difference between both groups. however, the mean level of hematocrit was increase (Table 6). It means that giving Moringa leaf extract capsules has the same ability as iron supplement to increase hematocrit levels in the blood. This study has limitation, such as the sample for pilot study is still small, further study is needed to evaluate the effect of moringa leaf extract capsules in broad community.

CONCLUSION:

This study showed that Moringa leaf extract capsules contain flavonoids, phenolics, alkaloids, tannins, saponins, steroids and terpenoids. It also showed total phenolic was 13.47% and a total flavonoid was 4.17%. The formula of Moringa leaf extract capsule exhibited good physical properties and not toxic based on the BSLT evaluation. This study also showed that Moringa leaf extract capsules had the same ability as iron supplements to increase hemoglobin levels up to 28.84% and hematocrit levels up to 22.99%.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors would like to thank Poltekkes Kemenkes Banjarmasin for their kind support during study and all other lab studies.

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Received on 13.09.2023 Modified on 08.12.2023

Research J. Pharm. and Tech 2024; 17(7):3375-3380.

DOI: 10.52711/0974-360X.2024.00527