INTRODUCTION:

Moringa plant (Moringa oleifera) belongs to the family of Moringaceae.¹ Because of all parts of this plant such as leaves, fruits, flowers, and roots can be used as food and traditional medicine,^2,3,4the leaves are rich in minerals, vitamins and other essential phytochemicals⁵so that it is very interesting for researchers. The Moringa tree can be grown in a home garden, and it has the ability to with stand long‐term drought conditions⁶. In addition, moringa plants can grow in tropical and subtropical areas around the world⁷including in Indonesia. Moringa plants are plants that do not know the seasons, so they are easy to obtain, and can thrive in almost all parts of Indonesia, including on the island of Lombok, West Nusa Tenggara province. Moringa become a district cross-sector program in NTB province and it formed into "Klorization Program" which the implementation regulated byRegent’s Regulation No. 80 of 2017concerning about the Moringa conservation program with the theme "Gemari Kelor" (the movement of community to plant and preserve moringa).

The parts of the Moringa oleifera plants that often used in traditional medication are the roots, seeds, leaves, and bark which are efficacious as antioxidants and as a treatment for various diseases such as diabetes mellitus ^8,9,10. Moringa leaves which are part of this plant provide several health benefits, including properties, antibacterial, anti-cancer, anti-inflammatory and anti-diabetic caused by the high content of phytochemicals, carotenoids, and glucosinolates from Moringa leaves.¹¹Moringa oleifera leaf powder has been found to have most of the essential nutrients needed for good health.⁶

Diabetes mellitus is a group of metabolic changes which characterized by hyperglycemia due to the defects of insulin secretion, insulin action, or both of them.^12,13 Diabetes is a chronic incurable disease and related to the significant morbidity and mortality. Although there are various approaches to reduce the adverse effects of diabetes and its secondary complications, but herbal formulations are preferred because of their lower side effects and low cost.¹². One of the popular herbs is Moringa oleifera Lam which called the "miracle tree", it is a monogeneric plant that is rich in minerals, carbohydrates, protein, fat, moisture content, rough fiber, and ash. Moringa oleifera contain a source of polyphenolic compounds such as flavonoids and phenolic acids.⁸ and also contains of zeatin, quercetin, beta-sitosterol and kaempferol^7,14.Become a researcher's question, does the Moringa plant that thrives on the island of Lombok, NTB province and becomes a cross-sectoral program, contain phytochemical compounds and have antioxidant activity ? To answer this question, it is necessary to conduct research that explores the content of phytochemical compounds and antioxidant activities of local Moringa leaves from the island of Lombok, West Nusa Tenggara province, Indonesia and predicts its role as a therapeutic candidate for diabetes mellitus disease.

This study aims to identify phytochemical compounds and analyze the antioxidant activity of Moringa leaf powder (Moringa oleifera) from the island of Lombok and predict its role as diabetes mellitus therapy.

This research is hoped can be used as a "data base" in facilitating the researcher’s before conducting laboratory research or clinical trials.

MATERIALS AND METHODS:

In-silico test.

In silico test conducted at Indonesian Bioinformatics and Biomolecular Services (INBIO-Indonesia) Malang, registered as global science company No.03/CV/I/2017. The test carried out in order to determine the interaction between a compound and the target molecule, which visualized by computational methods and can be used to determine the pharmacophore of a compound^15.

The compounds contained in Moringa leaves were obtained from the PhytoChem webserver, then predicted for their potential as antidiabetics through the analysis of Pa (Probability to be Active) with the Pass Server¹⁶ approach. Pa is a value that describes the ability of a compound in certain biological processes.The higher value of Pa, the more potential the compound. The approach used by the Pass Server is a structural approach which compare the inputted compounds with proven compounds that have certain activities. The higher value of Pa, the more similar the structure and function of the compound, when Pa has a value > 0.7 means that the similarity of the input compound to the database is high and most likely has biological activity if it tested in the laboratory, and when the Pa value is more than 0.3 and less than 0.7 means that the compound has the potential as a certain bioactivity computationally because there is little similarity in structure with the compound that has been recorded in the database.

To determine the interaction between proteins that play a role in diabetes and proteins targeted by Moringa leaves, protein interaction analysis performed by using the STRING DB¹⁷.

Manufacture of Moringa Leaf Powder (Moringa oleifera):

Materials:

The Moringa leaves (Moringa oleifera) obtained from Mataram City, Lombok Island, West Nusa Tenggara Province, Indonesia, and have been determined at the Biology Laboratory, Mataram university, with the certificate of identification No.35/UN18.7/LBL/2020. The results of the determination stated that the scientific/ Latin name of Moringa is Moringa oleifera.

Procedure for making Moringa leaf powder (Moringa oleifera):

The initial stage that is done before making the powder is to determined the Moringa leaves (Moringa oleifera), which aims to establish the correctness of the Moringa leaves to be used.

· The leaf picking from farmland

· The selection of Moringa leaves to be powdered is an adult (slightly old) Moringa leaf with a characteristic dark green Moringa leaf, ovoid in shape with flat leaf edges and small small sizes arranged compound in one stalk, the texture is thin limp and fresh.

· The Sorting process (Moringa leaves, separated from the twigs and stalks, and selected, leaves that are yellow, white-spotted, young or damaged are separated and removed).

· Weighing fresh Moringa leaves using branded food scales Oxone OX-311

· Washing Moringa leaves to remove dirt and dust using clean running water and when washing is not allowed

· To knead leaves.

· Washed leaves are then drained so that the water that is still attached to the leaves can completely disappear,

· Then drain using a special filter at a slope position of 90⁰to speed up the drying process.

· Drying Moringa leaves using an oven with a temperature of 40⁰C for 24 hours.

· After the leaves are dry, then weighing is carried out using a digital balance, namely the Ohause brand micro

· Analytical balance with an accuracy of 0.001 mg.

· Dried Moringa leaves are then ground using a blender and sifted using a 104 mesh sieve to produce leaf powder and separate the grains that are still rough.

· The moringa leaf powder produced is a raw material that is ready to be used, then packaged in laminated aluminum foil packaging thickness of 125 microns to be stored as stock.

Phytochemical Test:

Phytochemical test aims to determine the class of compounds contained in Moringa leaf powder (Moringa oleifera) from the Lombok island, Indonesia. Phytochemical tests were carried out qualitatively and quantitatively.

Phytochemical qualitative test of Moringa leaf powder:

Phytochemical qualitative test of Moringa leaf powder was carried out at the integrated research Laboratory of the Faculty of Mathematics and Natural Sciences, (FMIPA) Udayana University, Bali.

Phytochemical quantitative test of Moringa leaf powder:

Quantitative phytochemical tests (polyphenols, flavonoids and tannins) of Moringa leaf powder carried out by applying the spectrophotometric method at the integrated research Laboratory of the Faculty of Mathematics and Natural Sciences, (FMIPA) Udayana University, Bali, and quercetin by applying the spectrophotometric method at the Immunobiology Laboratory of FMIPA, University of Mataram.

Test of antioxidant capacity and antioxidant activity:

Testing of antioxidant capacity is a parameter that can describe the percentage of a food ingredient's ability to inhibit free radicals. The DPPH (1,1-diphenyl-2-picryl hydrazyl) method was used for the determination of in vitro antioxidant activity of Moringa olifera extract^4,18. Antioxidant capacity and activity tests were carried out at the Integrated Research Laboratory of the Faculty of Mathematics and Natural Sciences, Udayana University, Bali.

RESULTS:

In-silico test:

The compounds in Moringa predicted to have high potential as anti-inflammatory (0.707), this role is also supported by the potential of Moringa as an antioxidant (0.839) to bind oxidative stress. Thus, it is necessary to do further analysis in the form of interactions protein to determine the role of Moringa as an antidiabetic

Based on the analysis by using Pass Server, it is predicted that Moringa leaves can be used for the treatment of diabetes, because they have a high role as anti-inflammatory and antioxidant

String DB Interaction:

Figure 2. Analysis of protein interactions that play a role in diabetes and proteins targeted by Moringa leaves using STRING DB the protein interactions related to diabetes are RBP4 = Retinol Binding Protein4; IRS1 = Insulin Reseptor Substrat 1; DPP4 = Inhibitor dipeptidyl peptidase 4; SLC16A1 = solute carrier family 16 member 1, that play a role in the regulatory activity of insulin secretion, IRS1; SRC, INS have a role as a positive regulation on insulin receptor signaling and SLC16A1, INSR, INS, IRS1, RBP4 that play a role in the activity of glucose homeostasis.

Table 1. Results of Target Protein Analysis

Aktivitas	Indikator Warna	Protein
Regulation of Insulin Secretion	Green	RBP4,IRS1, DPP4, SLC16A1
Positive Regulation of Insulin Receptor Signaling Pathway	Red	IRS1,SRC, INS
Glucose Homeostatis	Yellow	SLC16A1, INSR, INS, IRS1, RBP4
Insulin Receptor Signaling Pathway	Blue	IGF1R, INSR, INS

Note: Bold letters are proteins targeted by Moringa leaves and are associated with diabetes, normal letters are input proteins associated with diabetes. Moringa leaves (Moringa oleifera) can target proteins RBP4 =Retinol Binding Protein 4; IRS-1= Insulin Reseptor

Substrat 1; SLC16A1 = solute carrier family 16 member 1; and SRC kinase.

Stitch Interaction:

Figure 3. Interaction analysis of Moringa leaf compounds (Moringa oleifera) using the STITCH program.

Based on the analysis by using the STITCH program,quercetin is one of the ingredients in Moringa leaves has a role to influence the regulation of the expression of Insulin Receptor Substrate (IRS)1 which plays an important role in pathway of insulin signaling through the receptor tyrosine kinase.^19,20 Therefore the IRS-1 gene is a candidate gene for type 2 diabetes because of its role in determining susceptibility to traits related to type 2 diabetes. In addition, ascorbic acid also has a role to interact with DMT1 although the type of interaction has not known yet.

Moringa Plant (Moringa Oleifera) and Moringa leaf powder

Figure 4. Moringa plant from Mataram City, Lombok island and moringa leaf powder. A. Moringa plant (Moringa oleifera) from

The city of Mataram, Lombok island, with characteristic compound leaves, long-stemmed, leaf blade when young is light green, after adulthood dark green, leaf blades are ovoid, limp thin, blunt leaf tips and bases, (obtusus), flat edge, pinnate, smooth upper and lower surfaces. B. Moringa leaves C. Moringa leaf powder. D. Moringa leaf powder in packaging

Test results of antioxidant capacity and antioxidant activity

Table 4. Analysis of antioxidant capacity and antioxidant activity

S. No	Parameter	Unit	Results
			Average	SD
1	Antioxidant capacity	mgGAEAC/L	1247,02	39,29
2	IC50	ppm	1312,99	10,04

The antioxidant activity test is expressed as percent inhibition (% inhibition), the value of Inhibition Concentration 50% (IC50) is determined, namely the concentration of the sample that can reduce DPPH free radicals by 50% using the equation y=ax+b

DISCUSSION:

Moringa oleifera commonly called as Drumstick tree is a multipurpose tree and it is widely present in in various countries such as India, Asia, Africa, etc ²¹including in Indonesia.In Indonesia, Moringa (Moringa oleifera) spreads from Java, Sumatra, Kalimantan, Sulawesi, East Nusa Tenggara and West Nusa Tenggara.

The Moringa leaves (Moringa oleifera) used in this study taken from trees located in Mataram city, Lombok Island, NTB Province. The leaves are processed into powder according to the procedure to adjust physicochemical properties such as carbon nanotubes (CNTs), through the functionalization of CNTs, it is conceivable to adjust their physicochemical properties, augmenting their simplicity of dispersion, control, and processability.²²if it is wrong in processing, then the Moringa leaves will become brownish-yellow and even grow mold, as a result of too long drying, high humidity due to poor air flow or low room temperature. Good dried Moringa leaves are green, completely dry (when kneaded it will crumble or moisture content 5%) and without petioles. Moringa leaf powder in this study had a moisture content of 5%. This process aims to ensure the quality of Moringa leaf powder remains fresh with longer shelf life where Moringa leaf powder can be stored at home for up to 6 months under recommended storage conditions and maintains the nutritional value and chemical compounds contained in it. It is related in pharmaceutical applications need to be characterized extensively^23.

In silico analysis is the early prediction whether a compound has contained expected efficacy, in order to be quicker to find new compounds and whether this research can be continued to be developed. Based on the analysis by using Pass Server, Moringa leaves predicted to be anti-inflammatory and antioxidant^.7 In silico test by using the STITCH program, showed that moringa oleifera contains of quercetin compounds. Based on the results of the research in the laboratory by using the spectrophotometric method, it was proven that Moringa leaf powder of Lombok island, not only contains quercetin but also polyphenols, flavonoids and tannins (table 3), and qualitatively, Moringa leaf powder is positive for alkaloids, polyphenols, flavonoids, tannins, and phenols (table 2). This study is in line with other studies which proved that moringa oleifera leaves contain the main flavonoid compounds, such as myricetin, quercetin and kaempferol with each concentrations of 5.8, 0.207 and 7.57mg/g,^24,25,26

The most plentiful flavonoid compound in Moringa leaves is quercetin²⁴. The In silico test showed that Moringa leaves contain quercetin, as well as the results of quantitative phytochemical analysis which showed that moringa leaf powder of Lombok contained of 30.33 mg/kg quercetin. Quercetin has hypolipidemic, hypotensive, and anti-diabetic characters to obese Zucker rats with metabolic syndrome.^24,27 can protect insulin-producing pancreatic cells from Streptozotocin (STZ) induced oxidative stress and apoptosis in mice.^24,28. The results of that study are in line with the in silico test results which showed that the compounds in Moringa are predicted to have high potential asanti-inflammatory (0.707), this role is also supported by the potential of Moringa as an antioxidant (0.839) to bind oxidative stress.

Oxidative stress can be caused by the increasing of ROS production as a result of high blood glucose levels in diabetes, so that cause cellular changes and activating apoptosis inβ-cells. Both apoptosis and necroptosis have important roles in the development of diabetic complications and they can cause the injury to heart, retina, kidney, and nervous system.²⁹ ROS are provoked by hyperglycemia and/or hyperlipidemia in diabetic conditions which lead to the activation of the JNK pathway in pancreas cells, ROS and activation of the JNK path induce the translocation of nucleocytoplasm from PDX-1, which leads to reduce PDX-1 activity and insulin emphasis. Therefore, there is a possibility that the ROS and activation of the JNK path involved in cell dysfunction found in type 2 diabetes.³⁰Diabetes mellitus is a clinical syndrome with insufficient insulin secretion and abnormal glucose tolerance. It is a group of diseases which results in too much sugar in the blood.²¹Antioxidants are compounds that are able to inhibit the oxidation of other molecules, which work through the breaking free radical chains mechanism , detoxification and activating antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) including glutathione reduction (GSH)³¹. The test of antioxidant capacity and antioxidant activity of Moringa oleifera powder from Lombok Island showed that a very weak character with the value of IC50 > 200ppm. It is different with the results of the in silico test which showed that Moringa oleifera is potentially as an antioxidant named PA which has a value of > 0.7 (0.839). The character of Moringa antioxidant probably due to the presence of phenolic compounds and the kaempferol content which shows the antioxidant activity.³².

Polyphenol is a group of natural compounds that contained in plants, and based on the results of phytochemical screening found in Moringa leaf powder from Lombok island. Polyphenols act as antioxidant molecules which can control the free radicals that can damage cells and cause various diseases. Besides having an antioxidant effect, polyphenols are also able to reduce uncontrolled inflammation which can cause various chronic diseases such as diabetes mellitus. Based on the analysis by using the Pass Server in the in silico test, it is predicted that Moringa leaves are potential as anti-inflammatory (0.707) because they contained polyphenol compounds which have the ability to control free radicals and reduce inflammation, to control blood sugar levels, because in this case polyphenols can prevent the breakdown of starch (amylum) into simple sugars. Polyphenol compounds can also stimulate insulin secretion, so that blood sugar can stay stable.³³

Flavonoids have various types and can be found in the free form (aglycone) or bound as glycosides. Polymethoxy aglycones are non-polar, polyhydroxy aglycones are semi-polar, while flavonoid glycosides are polar because they contain a number of hydroxyl groups and sugars ^34,35 The results of phytochemical screening of flavonoid compounds in Moringa (Moringa oleifera) leaves. Flavonoid compounds are polyphenolic compounds with a core which consist of 15 carbon atoms, composed of two benzene ring groups which linked together into a linear chain that consist of 3 carbon atoms which function as free radical scavengers, inhibitors of hydrolysis and oxidative enzymes, and as anti-inflammatory³⁹. According to Shaat 2017 and Yassa 2014 that the antioxidant activity of Moringa leaf powder has a strong potential as an antidiabetic in streptozotocin-induced diabetic rats.^36,37The flavonoids like quercitin and phenolics have been attributed as antioxidants that bring about a scavenging effect on ROS. It can be hypothesized that the flavonoids in Moringa scavenge the ROS released from mitochondria, thereby protecting the beta cells and in turn keeping hyperglycemia under control ⁵

Fig. 5. Mechanism of high glucose leading to diabetes and the effect of moringa on progression of diabetes. The high glucose in blood enters glycolysis in the mitochondria of beta cells and forms reactive oxygen species. This then causes apoptosis of beta cells which in turn leads to decreased insulin secretion, hyperglycemia and finally Type-2 diabetes. However, the cell apoptosis of beta cells can be averted by the use of moringa. Moringa has antioxidants which combine with the reactive oxygen species and prevent cell damage and further consequences 5

Tannins are active compounds of secondary metabolites that can function as biological antioxidants³⁸. The tannin content in Moringa leaf powder from the island of Lombok is 306.10 mgTAE/100 g, so it will affect to the antioxidant activity. This is supported by results of in silico test which proven that Moringa leaves are predicted to have high potential as antioxidants (0.839), this potential due to Moringa leaves contain several phytochemical compounds such as polyphenols, flavonoids, tannins and quercetin.

CONCLUSION:

Moringa leaf powder from the island of Lombok contains of alkaloid compounds, polyphenols, flavonoids, tannins of penoldan and quercetin and have very weak antioxidant capacity and activity. Moringa leaves (Moringa oleifera) can target RBP4, SLC16A1, SRC and IRS1 protein related to insulin pathway which predicted to can used for therapy of diabetes mellitus.

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Received on 03.06.2022 Modified on 16.08.2022

Research J. Pharm. and Tech 2023; 16(5):2169-2176.

DOI: 10.52711/0974-360X.2023.00356

S. No	Parameter	Method	Indication positif	Result
1	Saponin	Alcohol Reagent	Foam formed	Negative
2	Alkaloid	Meyer Reagent	Turbid	Positive
3	Flavonoid	ALCl3 Reagent	Yellow	Positive
4	Tannin	Follin Dennis Reagent	Blue	Positive
5	Steroid	Kloroform and sulfuric acid Reagent	Red Ring	Negative
6	Triterpenoid	Acetic acid, kloroform and sulfuric acid reagent	Reddish	Negative
7	Phenol	Follin Chiocalteu Reagent	Blue	Positive

S. No	Parameter	Method	Unit	Result
				Average	SD
1	Polyphenol	Spectrophotometry	mg GAE/100 g	864,09	8,91
2	Flavonoid	Spectrophotometry	mg QE/100 g	649,97	5,59
3	Tanin	Spectrophotometry	mg TAE/100 g	306,10	5,34
4	Quercetin	Spectrophotometry	mg QE/100g	30,33	4,82