Role of Moringa oleifera seed consumption on the levels of glucose, serum adipokines and bone function markers in patients with type 2 diabetes mellitus

The current study was conducted to investigate the impact of consumption of Moringa oleifera seed on the levels of glucose, Bone function markers and adipokines, in diabetes males. This study involved (47) diabetes male patients aged (40-59) years that have been classi(cid:976)ied into (Group 1): diabetes male under the treatment of Moringa oleifera seed (n=15) and (Group 2) diabetes male under the treatment of synthetic drugs (n=32). In addition to (15) appar-ently healthy subjects as a control group. Diabetes male patients consumed every day the Moringa oleifera seed at dose of 70 mg (one teaspoon) for 6 months. Blood samples were collected from both group to determine serum lipid pro(cid:976)ile, adipokines, bone function markers. Results indicate that consumption of Moringa oleifera seed induced a remarkable decrease in the levels of glucose and HbA1c, with accompanying increases in the levels of leptin and adiponectin. Furthermore, the use of Moringa oleifera seed increased vitamin D and inorganic phosphorus levels with concomitant decreases in the levels of osteoprotegerin and serum total calcium. These results show that consumption of Moringa oleifera seeds has a hypoglycemic effect, improving bone function markers and increasing adipokine levels in diabetic patients by improving biochemical indicators.

The current study was conducted to investigate the impact of consumption of Moringa oleifera seed on the levels of glucose, Bone function markers and adipokines, in diabetes males. This study involved (47) diabetes male patients aged (40-59) years that have been classi ied into (Group 1): diabetes male under the treatment of Moringa oleifera seed (n=15) and (Group 2) diabetes male under the treatment of synthetic drugs (n=32). In addition to (15) apparently healthy subjects as a control group. Diabetes male patients consumed every day the Moringa oleifera seed at dose of 70 mg (one teaspoon) for 6 months. Blood samples were collected from both group to determine serum lipid pro ile, adipokines, bone function markers. Results indicate that consumption of Moringa oleifera seed induced a remarkable decrease in the levels of glucose and HbA1c, with accompanying increases in the levels of leptin and adiponectin. Furthermore, the use of Moringa oleifera seed increased vitamin D and inorganic phosphorus levels with concomitant decreases in the levels of osteoprotegerin and serum total calcium. These results show that consumption of Moringa oleifera seeds has a hypoglycemic effect, improving bone function markers and increasing adipokine levels in diabetic patients by improving biochemical indicators.

INTRODUCTION
Plants and their derivatives have been used for the treatment of DM for thousands of years and are often thought to be less toxic and less adverse than their synthetic equivalents. For this reason, medicinal plants have long been consumed as a therapeutic substance for diabetes mellitus worldwide. Medicinal herbs, including Moringa oleifera, have attracted attention for their curative effect and controlling of diabetes. The antihepatotoxicity, antidyslipidemic, and anti-in lammatory action of Moringa oleifera, as well as its capability to regulate essential hepatic enzymes and biochemical markers, were assessed in experimentally induced diabetic rats (Kumar et al., 2015).
Current glucose-lowering administration agents have a little disadvantage in comparison to synthetic agents. However, they have side effects like a disorder of the skin, haematological disorders and increased activities of hepatic enzymes. Despite the development of pharmacological agents to treat diabetes, the use of therapeutic plants is considered a complementary remedy for this disease. The herbal treatment methods used in the treatment of diseases have been transferred through gener-ations. Nowadays, in light of this information, it has been important to explore the healing effects of bioactive compounds (Ota and Ulrih, 2017).
The specialised agency of the United Nations responsible for international public health has proposed the progression of oral glucose-lowering agents from medicinal herbals as a natural herbal medicament to treat patients with diabetes attribute to their being cost-powerful and safe (Singh et al., 2008). Moringa oleifera arose from India and is now grown across the world. So, individuals in many economically developing countries have been consuming Moringa oleifera to treat and prevent the symptoms of diabetes for years. This is primarily due to its natural advantages. Moringa oleifera has been revealed to naturally improve the immune system, which usually becomes impaired in those who suffering from diabetes mellitus. Moringa oleifera has also been indicated to possess various essential anti-in lammatory effects. There are no adverse side effects linked with the utilisation of Moringa oleifera, considering that it is a safe, natural way for an individual to regulate their blood glucose and other complications correlated with diabetes.
Moringa oleifera (MO), is an Asian herb also called Drumstick tree which has high medicinal value and can be cultivated in the north and south regions of the world. It is usually grown and cultivated in Pakistan and is also known as Sohanjna. Maximum height of its tree is up to 10 m. Its leaves, seeds, pods and roots are also used in treating lung diseases, hypertension and skin infection. MO leaves contain vital proteins, essential amino acids, trace elements, phenolic compounds, antioxidants, bioactive compounds, tannins that making it ideal against speci ic health problems (Nasir et al., 2017).
It is also plentiful in vitamins and minerals that mostly resides in leave parts in the form of vitamin B6, provitamin A as β-carotene, magnesium, calcium (Bharali et al., 2003), phosphorous, potassium, vitamin A and D as well as various antioxidants as vitamin C and lavonoids (Mbikay, 2012). The antioxidant and antihyperglycemic activity of aqueous extract of Moringa oleifera leaves have emerged as a potent antihyperglycemic in streptozotocininduced diabetic albino rats (Al-Malki and Rabey, 2015).

Study population and study design
Group 1 Composed of 15 diabetic male patients (under the consumption of Moringa oleifera seed: 70 mg (one teaspoon) every day for six months).

Group 2
Composed of 32 diabetic male patients (under treatment with synthetic drugs).
Group 3 (Normal Control) Composed of 15 healthy males.

Sample collection and evaluation
Blood specimens were collected from the study subjects (patients and healthy individuals) after 10-12 hours of fasting. The samples were preserved in Gold-top serum separator tubes (SST) and EDTA tubes before centrifugation process. The specimens were centrifuged at 3500 rpm for 10 min, and the resulting serum and plasma samples were separated and preserved in Eppendorf tubes. After the collection of all samples were completed, the serum and plasma samples were dissolved to be ready for biochemical analyses.

Biochemical assays
The circulating glucose level was determined by the enzymatic colourimetric method, using BIO-LABO kit (France). Also, the HbA1c (Hemoglobin A1c) in whole blood was measured by the colourimetric method involving [ luorescence Immunoassay (FIA) using Boditech Medkit. The serum leptin was determined by sandwich enzyme-linked immunosorbent assay (ELISA) technique using the Human Leptin (LEP) ELISA kit manufactured by SunLong Biotech, China. Furthermore, the serum adiponectin was determined by sandwich enzymelinked immunosorbent assay (ELISA) technique using the human adiponectin (ADP) ELISA kit manufactured by SunLong Biotech, China. Serum Osteoprotegerin was estimated by sandwich enzymelinked immunosorbent assay (ELISA) technique using the Human Osteoprotegerin (OPG) ELISA kit manufactured by Sun Long Biotech, China. The serum 25-OH Vitamin D Concentration was assessed by solid-phase sequential enzyme immunoassay (EIA) technique using a kit made by Monobind, USA. The determination of serum inorganic phosphorous and serum calcium were performed by using the enzymatic colourimetric method using BIOLABO kit (France).

Statistical Analysis
SPSS version 21 and GraphPad prism version 8 computer programs were used for statistical data analysis. Analytical test results and Bar graphs were performed expressed as Mean±SE. Unpaired T-test (Man-Whitney U) test was used for comparing the study parameter means between patient and control groups. The observed results in the current study indicate that dietary supplementation of Moringa oleifera seed has a positive blood sugar effects by reducing FBG, which is high-risk health factor for diabetic patients. It has been revealed that a considerable decrease in blood glucose levels over some time in treated samples was caused by Moringa oleifera (MO) extract. This decrease may result from increased releasing of insulin (by the action of MO) from pancreatic β-cells (Ra iu and Luka, 2015). Thus, diminished in the current glucose level declares the capability of extract to minimise hyperglycemia. Increase in the fall of blood glucose over time may be due to the components in the extract, which remains in the body for a long time and prevents DM. The capability of the seed extract of moringa oleifera to remarkably decrease high glucose level may attribute to signi icant plant constituents seed like micronutrient and phytochemical. The most important phytochemical constituent of the Moringa oleifera extract that has been recorded is lavonoids, which has been further identi ied by both structure and functional relation as; lavans, lavanones, lavones, lavanols, lavanonols, and iso lavones. Bio lavonoids are best known for their multi-purpose biological activities, including its ability to act as glucose-lowering effects (Szkudelski, 2001). Also, the Moringa oleifera contains many potent antioxidant phytochemicals, especially quercetin and kaempferol. Kaempferol has been known to have glucose-lowering activities. Also, the mode of action could be either by an increase in the tissue utilisation of glucose by Moringa oleifera blocking hepatic biosynthesis of glucose from the non-carbohydrate substance or through absorption of glucose into the muscles and adipose tissues (El-Desouki et al., 2015). The effect of Moringa oleifera seed on glycosylated haemoglobin levels (HbA1c) is presented in Table 1. The majority of metabolic disorders has been reported to increase HbA1c level in diabetic animals and to decrease serum insulin. HbA1c is used to monitor glycemic control in diabetic patients and is considered to be an essential biomarker for the progression and improvement of chronic diabetic complications (Katare et al., 2018). In this study, the HbA1c levels in diabetic patients under the treatment of herbal (seed of Moringa oleifera) showed a signi icant decrease, and this may be due to the positive effect of the seed extract on glucose metabolism, resulting in improved glycemic control.
The results of the current study are in line with (Shanker et al., 2019), who reported that mean glucose and HbA1c levels were reduced signi icantly in diabetic patients who used Moringa oleifera seed. Plants with anti-diabetic activity employ various mechanisms in living organisms (increasing insulin sensitivity and secretion, stimulating regeneration in Langerhans islets and preventing free radicals). Previous animal studies reported that Moringa oleifera seed extract has a lowering effect on blood glucose levels in diabetic rats. The antihyperglycemic impact of Moringa oleifera may be caused by stimulating the releasing of insulin from β-cells via prevention of the formation of free radicals (Bamagous et al., 2018). Several polyphenols are found in Moringa oleifera. Amongst the most powerful are the lavonoids quercetin and kaempferol, caffeoylquinic acid. These compounds appear to converse anti-diabetic properties, acting as a competitive blocker of the sodium-glucose linked transporter type 1, thus decreasing the intestinal absorption of glucose (Vargas-Sánchez et al., 2019).
However, absorption of glucose involves other mechanisms such as the glucose transporter 2, which can be engaged towards small intestine mem-  (Kellett and Brot-Laroche, 2005). In type 2 diabetes, the capacity of the small intestine to receive glucose is increased, as a result of an elevation in the expression of glucose transporter type 2 and sodium-glucose linked transporter type 1 (Dyer et al., 2002). This forms a new problem on the diabetic patients, further complicated because of consumption of anti-diabetic drugs such as sulfonylureas, biguanides or thiazolidinediones, that have principle targets on organs other than the intestines (Meneses et al., 2015). Moringa oleifera has been studied as an antihyperglycemic agent due to its actions on the lowering of glucose concentration. One of the suggested mechanisms contributed is quercetin, as this substance can act as a blocker of glucose transporter type 2. However, it does not affect glucose transporter type 5 or sodium-glucose linked transporter type 1.
Nevertheless, quercetin has also been revealed to phosphorylate adenosine monophosphateactivated protein kinase, to elevation glucose uptake through stimulation of glucose transporter type 4 in skeletal muscle, and to diminishing the formation of glucose through negatively regulation of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase in the liver. Moringa oleifera aqueous leaf extract has been shown to block the activity of α-glucosidase, pancreatic α-amylase, and intestinal sucrose, involving anti-diabetic properties (Vargas-Sánchez et al., 2019). These preventative effects are the possible advantage of phenols, lavonoids, and tannins present in Moringa oleifera. A delay in digestion of carbohydrate, caused by the blocking of these enzymes, causes a diminish in post-prandial high glucose level and haemoglobin A1C (HbA1C). These preventative impacts of lavonoids, including quercetin and kaempferol, have been biochemically described due to presence of a 2,3-double bond and to an increase in the number of OH groups on the B ring, (Tadera et al., 2006). Besides, these compounds have been examined upon their protective function and reformative characteristic on pancreatic beta-cells, increasing insulin secretion and release (Latif et al., 2014). Quercetin causes insulin production through phosphorylation of extracellular signal-regulated kinase 1/2 pathway and protection of pancreatic beta-cells against oxidative stress (Youl et al., 2010).
Glucose lowering activity in the bioassay due to the presence of phytochemicals, that act as bioactive compounds with an antioxidant property that has been linked with a protective effect versus chronic regressive diseases. These glucose lowering impacts have been examined with doses of seed powder (50-100 mg/kg body weight) in diabetic patients, wherein diminishes in fasting blood sugar and circulating haemoglobin A1c compared with controls have been recorded (Olayaki et al., 2015). Previous researches proposes that kaempferol enhances glucose uptake via the PI3K and PKC pathways in the rat soleus muscle. Orally consumed kaempferol, remarkably diminished fasting blood glucose and serum HbA1c concentrations while ameliorating insulin insensitivity. Quercetin blocks the transport of fructose and glucose by glucose transporter type 2 in the brain and stimulates glucose transporter type 4 expression in skeletal muscle (Sherein et al., 2014). This could describe the af inity towards lower blood glucose concentrations in the diabetes group treated with Moringa oleifera compared to the positive control group. The glucose-lowering effect of Moringa oleifera leaf (8 g/day) by dietary administration in a period time of 40-days in type 2 diabetic patients in the age range (30-60) years of age without pharmacological treatment was examined and revealed a remarkably decreased glucose response in comparison to the patients not taken Moringa oleifera leaf (Kumari, 2010). Another study showed that a group of type 2 diabetic patients, with age range 40-58 years given Moringa oleifera leaf tablets/day for 90 days exhibited that the blood glucose response developed descendingly with time while HbA1C showed lowering direction but not remarkably (Ghiridhari et al., 2011).  Table 2 also showed a remarkable increase (P=0.0029) in mean serum level of adiponectin in diabetic males who were under the treatment of herbal (seed of Moringa oleifera) which was (3.08±1.09 ng/ml) as compared to diabetic patients who were under the treatment of synthetic drugs (2.05±0.22 ng/mL).

Comparison the effect of M. oleifera seeds with synthetic antidiabetic drugs on the levels of adipokines
The results in this study revealed a remarkable increase in serum leptin concentration in herbal treated patients when compared to patients which used synthetic anti-diabetic drugs. At the same time, a non-signi icant increase of adiponectin level was seen in herbal treated patients in comparison to patients which used synthetic anti-diabetic drugs. Body fat that is stored within the abdominal cavity is considered as the direct association between various metabolic disorder and obesity, such as type 2 diabetes, hypertension and atherosclerosis, which are classi ied under the clinical signs of the metabolic disorder. Previous work (Ahmed et al., 2014) established the bene it of ethanolic extract of Moringa oleifera in ameliorating obesity and hyperlipidemia take place in female obese rats after a long period of therapy with HCD. Also, Moringa oleifera exhibited a bene icial effect in improving high serum level of leptin and low level of adiponectin. The direct potential mechanism for the describing of the curative role of Moringa oleifera in ameliorating the reported metabolic disturbance is the nature of M. oleifera as an antioxidant substance. The hypoglycemic effect of Moringa oleifera due to presence of various active elements like vitamins, minerals, amino acids, carotenoids, alkaloids, and lavonoids and contains phenolic compounds (such as zeatin, quercetin, isoquercetin, kaempferol, apigenin and rutin) (Yassa and Tohamy, 2014). Several studies showed that in the fatty tissue of obese rats, the gene expression of leptin was elevated. In contrast, the gene expression of adiponectin was diminished compared to the expression observed in the control rats.
The treatment with Moringa oleifera extracts negatively-regulated leptin mRNA expression while it positively-regulated adiponectin mRNA expression. Reduction in the leptin gene expression upon medicament with M. oleifera provided the principle pathway in the ameliorating of adiposity. Previous researches proposed a two-way correlation between serum leptin level and leptin mRNA in fatty cells and adiposity level (de Queiroz et al., 2014;de Azevedo Melo Luvizotto et al., 2013). de Azevedo Melo Luvizotto et al. (2013) recorded that utili-sation of high-fat meals for an extended period resulted in increased expression of leptin which due to the elevated mass of adipose tissue (de Queiroz et al., 2014). In his study demonstrated that the hyperleptinemia as a result of leptin mRNA and protein positively-regulation enhanced rats to show a high index of adiposity after feeding a diet high in sucrose (de Queiroz et al., 2014).
Furthermore, positively-regulation of adiponectin mRNA exhibited amelioratingin the fatty cell of the obese rat. Adiponectin mRNA negatively-regulation is a symptom of fatty cell precipitation in obese rats and mice and a defect of insulin sensitivity (Choi et al., 2016). Additionally, Moringa oleifera might act as an insulin sensitizer as it act in a similar track to anti-resistin antibody, which promote insulinmediated uptake of glucose in a fatty cell (Ye et al., 2013). The additional signi icant role of positivelyregulation of adiponectin gene expression upon treatment with Moringa oleifera was the decrease in the abnormalities of metabolic pathway occurred in obese rats. Adiponectin is an antiatherogenic agent that reduce cardiovascular risk, which may be due to its anti-in lammatory properties. It blocks dysfunction of the endothelial cell, because it inhibits the expression receptors of low-density lipoprotein scavenger" on macrophages, thus decreasing low-density lipoprotein uptake and formation of plaque. Contrary to leptin, adiponectin ameliorates insulin sensitivity, and its level in serum is negatively proportional to adiposity concentration. Obese patients with a decreased level of adiponectin progressed pattern of leptin resistance followed by insulin insensitivity, hyperlipidemia and heart disease (Metwally et al., 2017).
Gene expression analysis supplied a mechanistic curative role of ethanolic extract of M. oleifera in the controlling of obesity and reduction of abnormalities of metabolic pathways that occurred in cardiac muscle, resulting from high cholesterol diet (HCD) in female rats. Moringa oleifera extract worked directly on the fat mass and improved the defect of mRNA expression of leptin and adiponectin genes. Thus, it exhibits ameliorating in body mass index, atherogenic hyperlipidemia and insulin resistance. Therefore, Moringa oleifera could provide potent phytotherapy in the ameliorating of high levels of cholesterol, hardening of arteries and type 2 diabetes but without complications.  The indings in the current research are in line with (Habib and Al-Moalem, 2018;Srikanta, 2011). Type II DM is a disorder of carbohydrate metabolism linked with several complications, including a defect in the process of healing of the bone fracture. Bone is considered as an important skeletal structure in the body, is in luenced by diabetic status, especially during the process of fracture healing. Bone is a tissue that undergoes frequent repairing and has a high magnitude for reformation. Disturbance in the balance between bone resorption and new bone formation leads to bone loss (He et al., 2004). Bone loss has also been linked with diabetes mellitus. Several studies have recorded that type 1 diabetes changes bone repaiting by decreasing the synthesis of new bone, causing weakness of bone. This has been described by a reduction in mineral density of the bone in humans and variation in the production of new bone in animal researches. Several studies have con irmed the ef icacy of medicinal plants in ameliorating bone health. The literature study of osteoporosis reveals that vitamin D, calcium and garlic oil, in ovary removed rats with low bone mass has been achieved by (Holick et al., 2008). From the previous studies, it can be seen that there are many medicinal plants for DM and fragile bone. Still, very little are having a positive impact both on carbohydrate as well as mineral metabolism. One of such plant is Moringa oleifera (MO), which has been labelled as a "wonder tree" as the advantages of MO is abundant: each part of the tree is advantageous in several ways. Moringa oleifera leaves are a great source of minerals (calcium, iron), protein and vitamins (especially Vitamin A, B and C). They are used to ighting malnutrition, hypertension, blindness, diabetes mellitus, anaemia, renal stones, to enhance lactation in nursing women, and as a disinfectant. Moringa oleifera plant has been con irmed to possess the strength of aginst bone damage. It has already been established that lavonoids content of Moringa oleifera (MO), have anti osteoclastic activity. As the lower is rich in these lavonoids, one can understand the potent effect of lower extract on this osteoclastic function marker.

Comparison the effect of M. oleifera seeds with synthetic antidiabetic drugs on the levels of bone function markers
It has been reported that Moringa oleifera is an excellent source of phenolic phytochemicals and variety of curative properties. Phenolic phytochemicals are now indicated to have a possible role in controlling various diseases associated with chronic oxidation; one of them is type 2 DMs (Patel et al., 2015;Farooq, 2012). Reported that powder of Moringa oleifera contains a suf icient amount of protein, minerals, phenols, vitamins and different phytonutrients. This making the Moringa oleifera to act as a therapeutic substance for various illnesses. Kane et al. (2017) and Gopalakrishnan et al. (2016) reported that Moringa oleifera is one of the most extravagant plant wellsprings of minerals, such as Calcium, Copper, Iron, Potassium, Magnesium, Manganese and Zinc. Both protective and therapeutic role of Moringa oleifera in ight-ing human illnesses due to their content of minerals. For example, calcium is a multi-purpose supplement essential to the body digestion, while de iciency of calcium causes loss of bone mass. In this way, Moringa oleifera is showed the best solution for the treatment of fragile porous bones (Habib and Al-Moalem, 2018).
Additionally, administration the ethanolic extract of Moringa oleifera seeds was observed to diminish the circulating concentration of mediators of in lammation, the volume of paw oedema, and to prevent cytotoxic T-lymphocyte cells that cause anterior pituicyte destruction, also to reduce the destruction of bone and cartilage erosion in the synovial joint, following to the progression of arthritis in rats. Furthermore, seeds of Moringa oleifera assist in treating joint in lammation, stiffness, arthritis, anti-in lammatory agents and cramp (Habib and Al-Moalem, 2018). Several studies have been published, describing the role of Moringa oleifera as a potent pharmaceutical agent, one of which was presenting its role in the protection bones from mass loss in rats with removed ovaries (Burali et al., 2010). It has been reported that Moringa oleifera having cell forming bone property, as it augmented all the indicators of osteoblast activity. Remarkable osteoblast activity of Moringa oleifera attribute to the presence of fruit and lower extract. In a study by Vali et al. (2007), it was revealed that bone formation in vitro due to the signi icant action of many lavonoids. Moringa oleifera plays a crucial role in stimulating osteoblastic cells because MO is rich in various lavonoids. It has been reported that Moringa oleifera fruit extract was shown to be having a signi icant effect on bone loss; it was also shown to be having a signi icant impact on calcium homeostasis (Patel, 2013). Furthermore, Rangrez et al. (2011) revealed that Moringa oleifera could inhibit the loss of bone mass in ovaries removed promote bone loss.

CONCLUSIONS
The data obtained from the present study have established the hypoglycemic effect of Moringa oleifera seed consumption by the improvement of the glucose & HbA1c levels. The current indings suggested utilising Moringa oleifera seeds as an excellent anti-osteoporotic activity. Furthermore, seeds of Moringa oleifera have ameliorating adipokine action.