Moringa oleifera from Cambodia Ameliorates Oxidative Stress,Hyperglycemia,and Kidney Dysfunction in Type 2 Diabetic Mice

Abstract

Recent reports have shown the antidiabetic effect of Moringa oleifera from various parts of the world. However, M. oleifera from Cambodia has never determined. Therefore, the aim of this study was to assess the antidiabetic effect of M. oleifera extract from Cambodia. The leaf ethanolic extract contained flavonoids (31.90 mg/mL), polyphenols (53.03 mg/mL), lycopene (0.042 mg/mL), and ß-carotene (0.170 mg/mL), and possessed 2,2-diphenyl-1-picrylhydrazyl, hydrogen peroxide, and hydroxyl radical scavenging activities of 92.40, 99.25, and 83.57 TE/μM at 1 mg/mL, respectively. Db/db mice were orally administered the leaf extract (150 mg/kg/day) for 5 weeks. M. oleifera treatment significantly ameliorated the altered fasting plasma glucose (from 483 to 312 mg/dL), triglyceride (from 42.12 to 23.00 mg/dL), and low-density lipoprotein cholesterol (from 107.21 to 64.25 mg/dL) compared to control group, and increased the insulin levels from 946 ± 92 to 1678 ± 268 pg/mL. The histopathological damage and expression levels of tumor necrosis factor-alpha, interleukin (IL)-1β, IL-6, cyclooxygenase-2, and inducible nitric oxide synthase in renal tissue decreased. These results indicate the potential antidiabetic benefits of M. oleifera ethanolic leaf extract.

Introduction

In many countries, diabetes mellitus (DM) is a common disease. Changing lifestyles, reduced physical activity, and widespread obesity contribute to the increasing number of patients with DM.¹ In addition, recent worldwide increases in DM incidence project that by 2030 there could be 530 million people with DM globally.^2,3 Hyperglycemia is the major cause of diabetic complications, including retinopathy and nephropathy.⁴ Increased oxidative stress, impaired antioxidant defense systems, and consequent lipid peroxidation are major participants in the development and progression of DM and its complications.⁵ Therapeutic management of DM with minimal side effects remains a clinical challenge. As medicinal plants are commonly cheaper, less toxic, and have fewer side effects compared to conventional drugs,⁶ there is a growing interest in their potential use as an alternative treatment for diabetes.

The impairment of carbohydrate, lipid, and protein metabolisms and defects in insulin signaling lead to hyperglycemia, a defining characteristic of DM, which is associated with oxidative stress.^7,8 Hyperglycemia increases the formation of reactive oxygen species (ROS) causing a decrease in the levels of antioxidants.⁷ In addition, oxidative stress plays an important role in the development of diabetes.⁸ Antioxidants scavenge free radicals and ROS, and protect the body from oxidative stress. Hence, drugs with both antioxidant and antidiabetic properties would be useful in the treatment of patients with diabetes.⁹

Moringa oleifera Lam, Moringaceae, is a highly nutrient-rich plant with exceptional medicinal properties.¹⁰ M. oleifera is cultivated in many tropical and subtropical countries of Asia and Africa,¹¹ and is a good source of proteins, β-carotene, vitamins A, B, C, and E, riboflavin, nicotinic acid, folic acid, pyridoxine, amino acids, minerals, and various phenolic compounds.^12,13 Previous reports have shown the potential of M. oleifera grown in India, Egypt, and other parts of the world as a source of antidiabetic agents.^11,14

–17 In addition, the antioxidant properties of M. oleifera also have been reported recently.^18
–20 However, the in vitro and in vivo antidiabetic capacities of M. oleifera from Cambodia have never been determined. Geographic location was found to influence the biological activity of this species.¹⁷ Therefore, in this study, we investigated the antidiabetic activity of M. oleifera leaf from Cambodia in vitro and in vivo, to ascertain the potential of the plant material from this locale for use in the treatment of patients with diabetes.

Materials and Methods

Materials

All chemicals including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2-deoxy-D-ribose, gallic acid, ethylenediaminetetraacetic acid (EDTA), Folin-Ciocalteu's phenol reagent, hydrogen peroxide, 2,2-azino-bis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS), thiobarbituric acid (TBA), trichloroacetic acid (TCA), hydrogen peroxide, peroxidase, sodium carbonate, aluminum chloride (AlCl₃) and metformin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals and reagents used were of analytical grade and commercially available.

Plant collection

Fresh M. oleifera leaves, stems, and seeds were collected in May 2013 from Cambodia, between latitudes 10° and 15°N and longitudes 102° and 108°E. The leaves, stems, and seeds were dried at 65°C for 12 h.

Plant extract preparation

For aqueous extracts, 10 g plant samples (leaves, stems, and seeds, separately) were added to 100 mL of boiling distilled water and subjected to extraction for 1 h. For ethanolic extracts, 10 g samples were added to 100 mL of 70% ethanol and extracted for 2 h, and repeated thrice. Extracts were filtered and lyophilized in a freeze dryer for 5 days.²¹

DPPH radical scavenging activity

The DPPH scavenging activity of each extract was measured according to a slightly modified version of the method of Blois.²² DPPH solutions (1.5 × 10⁻⁴ M, 100 μL) were mixed with and without each extract (100 μL), and the mixtures were incubated at room temperature for 30 min. After standing for 30 min, absorbance was recorded at 540 nm using a microplate reader. The scavenging activity was calculated as a percentage using the following equation:

\documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$${ \rm{Inhibition }} \, \left( \% \right) = \left( { \left( {{{ \rm{A}}_{{\, \rm{control}}}} - {{ \rm{A}}_{{\, \rm{sample}}}}} \right) / {{ \rm{A}}_{{\, \rm{control}}}}} \right) \times 100 \ { \rm{where}} {{ \rm{A}}_{{ \rm{control}}}} \ { \rm{and}} \ {{ \rm{A}}_{{ \rm{sample}}}}$$ \end{document} are the absorbance of the reaction mixture without and with the sample, respectively, at 517 nm.

Hydrogen peroxide radical scavenging activity

The hydrogen peroxide scavenging activity was determined according to the method of Müller.²³ One hundred microliters of 0.1 M phosphate buffer (pH 5.0) was mixed with each extract in a 96-microwell plate. Hydrogen peroxide (20 μL) was added to the mixture and incubated at 37°C for 5 min. After the incubation, 30 μL of 1.25 mM ABTS and 30 μL of peroxidase (1 unit/mL) were added to the mixture, and then incubated at 37°C for 10 min. Absorbance was recorded at 405 nm by a microplate reader and the percentage of scavenging activity was calculated using the following equation: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm{Inhibition}} \ \left( \% \right) = \left( { \left( { \left( {{{ \rm{A}}_{{\, \rm{control}}}} - {{ \rm{A}}_{{\, \rm{sample}}}}} \right) / {{ \rm{A}}_{{\, \rm{control}}}}} \right) } \right) \times 100 \end{align*} \end{document}

where A_control and A_sample are the absorbance of the reaction mixture without and with sample, respectively, at 405 nm.

Hydroxyl radical scavenging activity

The hydroxyl radical scavenging activity of extracts was determined according to the method of Chung et al. ²⁴ Hydroxyl radical was generated by the Fenton reaction in the presence of FeSO₄. A reaction mixture containing 0.1 mL of 10 mM FeSO₄, 10 mM EDTA, and 10 mM 2-deoxyribose was mixed with 0.1 mL of the extract, after which 0.1 M phosphate buffer (pH 7.4) was added into the reaction mixture until the total volume reached 0.9 mL. Subsequently, 0.1 mL of 10 mM H₂O₂ was added to the reaction mixture and incubated at 37°C for 4 h. After incubation, 2.8% TCA and 1.0% TBA, 0.5 mL each, were added and the mixture was placed in a boiling water bath for 10 min. Absorbance was measured at 532 nm and the percentage of scavenging activity was calculated using the following equation: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}{ \rm{Inhibition}} \ \left( \% \right) = \left( { \left( {{{ \rm{A}}_{{\, \rm{control}}}} - {{ \rm{A}}_{{\, \rm{sample}}}}} \right) / {{ \rm{A}}_{{\, \rm{control}}}}} \right) \times 100 \end{align*} \end{document}

where A_control and A_sample are absorbance of the reaction mixture without and with sample, respectively, at 532 nm.

Fourier transform infrared spectroscopy

The Fourier transform infrared (FT-IR) spectrum was obtained using a FT-IR spectrophotometer Thermo-Nicolet Model 6700 (Thermo Scientific, USA), equipped with Smart Orbit (Diamond) ATR accessory and OMNIC 7.3 software, in the 6300–350 cm⁻¹ range at a resolution of 2 cm⁻¹ in transmission mode.

Determination of the total polyphenols content

The total phenolic content of the extract was determined with the Folin–Ciocalteu assay.²⁵ Samples (0.1 mL) of the extract in distilled water (1 mg/mL) were mixed with 50 μL of 50% Folin–Ciocalteu reagent, and 150 μL of 20% sodium carbonate (Na₂CO₃) was added. The solution was incubated at room temperature for 30 min. The absorbance of the reaction mixtures was measured at 760 nm. Gallic acid was used as the standard, and the total polyphenol content of the M. oleifera extract was expressed in milligram gallic acid equivalents (mg GAE/g extract).

Determination of the total flavonoid content

The total flavonoid content was estimated by the aluminum colorimetric method,²⁶ with catechin as the standard. Test samples were dissolved in distilled water, and 150 μL of the sample solution was then blended with 150 μL of 2% AlCl₃. After incubating the mixture for 10 min at room temperature, the absorbance of the supernatant was measured at 510 nm with a spectrophotometer. The total flavonoid content was expressed as catechin equivalents in milligrams per gram extract (mg CE/g extract).

β-Carotene and lycopene assay

β-Carotene and lycopene were determined according to the method of Nagata and Yamashita.²⁷ The dried extract (100 mg) was vigorously shaken with 10 mL of acetone–hexane mixture (4: 6) for 1 min and filtered through Whatman No. 4 filter paper. The absorbance of the filtrate was measured at 453, 505, and 663 nm. Contents of β-carotene and lycopene were calculated according to the following equations: lycopene (mg/100 mL) = −0.0458_A663 + 0.372_A505 − 0.0806_A453 and β-carotene (mg/100 mL) = 0.216_A663 − 0.304_A505 + 0.452_A453. The results are expressed as milligrams per gram extract.

Animal experiments

C57BLKS/J Iar-+Lepr^db/+Ledpr^db (29–30 g) and C57BLKS/J Iar-m⁺/Lepr^db (18–20 g) 7-week-old male mice were used in this work. Mice were provided by Samtako Bio Co (Osan, Korea). They were maintained in an air-conditioned room (20–25°C) and subjected to a 12-h day light/12-h darkness cycle with free access to food and water. C57BLKS/J Iar-+Lepr^db/+Ledpr^db were divided into three groups as follows: Group 1 served as the sham control group and were administered phosphate-buffered saline; Group 2 received 150 mg/kg body weight of the M. oleifera extract; and Group 3 received 150 mg/kg body weight of metformin (standard drug). C57BLKS/J Iar-m⁺/Lepr^db mice received 150 mg/kg of the leaf extract. Each group consisted of eight animals. There are several references for the dosage,^15,28

–31 and we decided to use a relatively low dosage among them. The extract was administered by gastric intubation. At the end of application, the gavage tube was left in place for several seconds to avoid regurgitation and assure supplying the total calculated dose. The extract was given once daily at a fixed time for the whole 5-week period of the experiment. At the end of the experiment, all mice were deprived of food overnight. Blood samples were collected from the heart. The blood and tissue samples were stored in a deep freezer (−80°C). All animal care procedures and experiments were approved by the Institutional Animal Care and Use Committee of Konkuk University (KU13073).

Blood glucose monitoring

Blood samples were obtained from the tail vein of the mice. Blood samples (5 μL) were added to glucose test strips as soon as possible. After about 3–5 sec, the results were read from the blood glucose monitor. The blood glucose estimation was done using a blood glucose monitoring kit (Infopia 21, Anyang, Korea). The estimated blood glucose levels were in a similar range between 250 and 600 mg/dL by Waterman et al. ³²

Serum biochemical parameters

Serum was obtained by centrifugation at 3500 g for 15 min at 4°C. The insulin and high-molecular-weight adiponectin levels were determined using an ELISA kit (Shibayagi Co., Ltd., Gunma, Japan). The content of triglycerides, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) was determined using selective elimination, with a kit from Wako Pure Chemical Industries, Ltd. (Chuo-ku, Osaka, Japan).

RNA isolation and mRNA expression analysis

For the reverse transcription polymerase chain reaction, the total cellular RNA was isolated from cells using TRIzol according to the manufacturer's protocol. The first-strand complementary DNA (cDNA) was synthesized using Superscript II reverse transcriptase (Invitrogen, USA). Polymerase chain reaction was performed as previously described, with exception of primer sets of the following primers: tumor necrosis factor-alpha (TNF-α) (s 5′-ACC AGG AGA GAA AGT CAA CCT C-3′; as 5′-GGA CTC CGC AAA GTC TAA GT-3′); interleukin (IL)-1β (s 5′- TCT GTG ACT CAT GGG ATG AT-3′; as 5′-TAT TTT TGT CGT TGC TTG GTT-3′); IL-6 (s 5′-GAG ACT TCC ATC CAG TTG C-3′; as 5′-CTC TTT TCT CAT TTC CAC GA-3′); cyclooxygenase-2 (COX-2) (s 5′-CCC CTC TCT ACG CAT TCT AT-3′; as 5′-AGG TCG TTT GTT GGG ATT AT-3′): and inducible nitric oxide synthase (iNOS) (s 5′-ATC ATG AAC CCC AAG AGT TT-3′; as 5′-AGA GTG AGC TGG TAG GTT CC-3′); and GAPDH (s 5′-GGT TGT CTC CTG CGA CTT CA-3′; as 5′-TAG GGC CTC TCT TGC TCA GT-3′) was used as internal control.

Histological analysis

The kidneys of the mice were fixed with 10% paraformaldehyde and embedded in paraffin. The paraffin blocks were sliced into 5-μm-thick sections, deparaffinized, and stained with hematoxylin and eosin.

Statistical analysis

All results are expressed as the mean ± standard deviation. In vitro data are representative of three independent experiments. Statistical analyses were performed using SAS statistical software (SAS Institute, USA). Treatment effects were evaluated by one-way analysis of variance, followed by Dunnett's multiple range tests for in vitro experiments, and post hoc Student's t-test for in vivo experiments. The cutoff P < .05 was used to indicate significance.

Results

The DPPH radical scavenging activity was the highest for the ethanolic leaf extract (92.40 μM TE/mg) and lowest for the ethanolic seed extract (14.30 μM TE/mg) (Fig. 1A). For hydrogen peroxide radical scavenging, all fractions exhibited a good percentage inhibition (Fig. 1B), and the ethanolic leaf extract showed the strongest potential (99.25 μM TE/mg). The hydroxyl radical scavenging activity was the highest for the ethanolic leaf extract (83.57 μM TE/mg) (Fig. 1C). As the ethanolic leaf extract exhibited the highest antioxidant activity in all radical scavenging tests, it was selected for further experiments.

FIG. 1.

Antioxidant activity of the Moringa oleifera extract. (A) DPPH radical scavenging activity; (B) hydrogen peroxide radical scavenging activity; (C) hydroxyl radical scavenging activity. *The highest activity of groups shown at P < .05. , 125 μg/mL; , 250 μg/mL; , 500 μg/mL; , 1000 μg/mL. DPPH, 2,2-diphenyl-1-picrylhydrazyl.

The phytochemical composition of the ethanolic leaf extract was quantified using biochemical assays. Polyphenol, flavonoid, lycopene, and ß-carotene contents are shown in Table 1. Polyphenols and flavonoids, the major components found in the extract, were 53.03 and 31.90 mg/g; the lycopene and ß-carotene contents were 0.042 and 0.170 mg/g.

Table 1.

Phytochemical Composition of the Ethanolic Extract of Moringa oleifera Leaf

	The ethanolic leaf extract
Flavonoids (mg/g)	31.900 ± 0.890
Polyphenols (mg/g)	53.030 ± 3.250
Lycopene (mg/g)	0.040 ± 0.002
ß-carotene (mg/g)	0.170 ± 0.002

The FT-IR spectrum of the M. oleifera leaf extract is shown in Figure 2. The strong broad band at 3265 cm⁻¹ was attributed to the presence of OH stretching in hydrogen bonds and N–H vibration. The signal at 2928 cm⁻¹ is attributable to C–H stretching vibrations, whereas the absorption peak in the region of 1651 cm⁻¹ was attributable to associated water. The absorption peak at 1543 cm⁻¹ showed the presence of phenolic C = C stretching vibrations. The peak at 1408 cm⁻¹ is associated with the typical stretching frequencies of OH groups from phenolic compounds. The peak at 1240 cm⁻¹ indicated aromatic (C–O–C) frequencies and overlapped with absorption frequency of C–N stretching. Peaks at 1021, 984, and 834 cm⁻¹ correlated to C–O–C linkages of sugar components remaining in the extract.^33,34

FIG. 2.

Fourier transform infrared spectrum of the ethanolic extract of M. oleifera leaf. Arrows indicate the wavenumber (cm⁻¹).

Blood glucose levels were measured once weekly in diabetic and normal mice. The results are summarized in Figure 3A. Diabetic mice showed significant differences in blood glucose levels compared to normal mice (P < .05). The groups treated with the M. oleifera extract showed significant decreases in blood glucose concentrations from day 14 to 35 after oral administration. Insulin levels were significantly improved by the extract (Fig. 3B). In addition, repeated administration of the leaf extract significantly decreased triglyceride (TG) and LDL-C levels (Fig. 3C, D).

FIG. 3.

Effect of the leaf extract on serum biochemical parameters. (A) Blood glucose concentrations; (B) insulin concentrations (pg/mL) of the studied groups; (C) triglyceride concentrations (mg/dL) of the studied groups; (D) LDL-C concentrations (mg/dL) of the studied groups. Diabetes mellitus + phosphate-buffered saline (DM + PBS); DM + M. oleifera leaf extract (DM + Moringa); DM + metformin (DM + metformin). *Significant difference from DM + PBS group shown at P < .05. LDL-C, low-density lipoprotein cholesterol.

We also investigated the expression of inflammation-related genes. Initially, the mRNA expression levels of TNF-α, IL-1β, IL-6, COX-2, and iNOS from kidney tissues were estimated. As shown in Figure 4, treatment of kidney tissues with the leaf extract and metformin significantly decreased the expression of TNF-α, IL-1β, IL-6, COX-2, and iNOS. Histopathology examination of kidneys of diabetic animals showed inflammation in blood vessels, increase in the thickness of Bowman's capsules, and decrease in the size of the glomerulus. Leaf extract treatment improved kidney histopathology showing reduced inflammation in the blood vessels, and the glomerulus size was similar to the normal group (Fig. 5).

FIG. 4.

The expression of inflammation genes measured by using RT-PCR. GAPDH was used as control. The results were similar in three independent experiments. RT-PCR, reverse transcription polymerase chain reaction.

FIG. 5.

Effect of M. oleifera extract on kidneys in different mouse groups. Black arrow: average size of glomerulus.

Discussion

Overproduction of free radicals plays a role in long-term complications of diabetes.³⁵ Therefore, we evaluated the antioxidant activity of various parts of M. oleifera from Cambodia extracts to select one part for further study.

As more than one mechanism may be involved in the antioxidant activity of M. oleifera extracts from Cambodia, three methods (DPPH, hydrogen peroxide, and hydroxyl radical scavenging activities) were used to valuate different aspects of the activity. The DPPH radical scavenging activity is an established assay for determining the antioxidant activity of herbal extract and phytochemicals and is widely used to evaluate changes in parameters of oxidative stress in diabetes.¹⁸ Sreelatha and Padma³⁶ demonstrated that the methanolic extract of M. oleifera leaf from Singapore significantly reduced DPPH radicals, although the activity was lower than our observed results. Difference in activities could be caused by the different habitats of the plants used.³⁶ Although hydrogen peroxide itself is not very reactive, it may convert into more reactive species, such as singlet oxygen and hydroxyl radicals formed by the Fenton reaction, subsequently initiating lipid peroxidation or cytotoxicity.³⁷ Determining the hydrogen +peroxide scavenging activity is known to be a useful method for determining the ability of antioxidants to decrease the level of pro-oxidants.³⁸ Our results are similar to the findings of Shih et al.³⁹ showing that methanolic leaf extract of M. oleifera from Taiwan exhibits the greatest hydrogen peroxide scavenging activity. Among the ROS, hydroxyl radicals show the highest chemical reactivity, reacting easily with amino acids, DNA, and membrane components. Removal of hydroxyl radicals is probably one of the most effective defensive mechanisms against various diseases.⁴⁰ The ethanolic leaf extract demonstrated the highest antioxidant activity in all radical scavenging tests; it was therefore selected for further experiments.

Phytochemicals have great potential for balancing metabolic disturbances. Several phytomolecules, including polyphenols, flavonoids, lycopene, and ß-carotene obtained from various plant sources, have been reported as potent hypoglycemic and antihyperglycemic agents.¹⁸ Flavonoids are a heterogeneous group of ubiquitous plant polyphenols that exhibit a variety of pharmacological activities, including antiatherogenic and antihyperglycemic effects, protection against lipoprotein oxidation and platelet aggregation, and vascular reactivity.^41,42 A high content of total phenolic compounds and flavonoids may have a significant role in regulating DM and its complications due to the protective effect of these compounds on the pancreas and other essential organs. Flavonoid-rich plant extracts have been reported to show hypoglycemic and antidiabetic activity.^43,44 M. oleifera leaf extract is a rich source of flavonoids and phenolic compounds.^17,45 Our results found significantly higher flavonoid content than that found in the leaf extract of M. oleifera from India.³⁶

DM is a metabolic disorder affecting carbohydrate, fat, and protein metabolism, followed by multiorgan dysfunction in the later stages.⁴⁶ M. oleifera leaf extract decreased serum glucose levels and increased insulin levels as shown in Figure 3A, B. Also, a lot of research in diabetes is carried out to find insulin substitutes, secretagogues, or sensitizers from synthetic or plant sources. Our results suggest that M. oleifera leaf extract may stimulate insulin secretion and decrease serum glucose levels. In addition, the dose of the M. oleifera from Cambodia used in this study is much lower compared to other studies.^26

–29 Moreover, repeated administration of the leaf extract significantly decreased TG levels. The observed effect may be caused by decreasing cholesterolgenesis and fatty acid synthesis through inhibition of pancreatic cholesterol esterase and pancreatic lipase, respectively.^47,48 Extracts of M. oleifera from Ethiopia lowered TG levels much more than ours (Fig. 3C), which could be attributable to different animals used in their study and different geographic location. The LDL-C level was significantly improved by the leaf extract treatment (Fig. 3D). However, there was no significant increase of HDL-C (data not shown), which was consistent with the investigation by Kumari.⁴⁹ Meanwhile, the pattern of the body weight changes and the food efficacy ratio (FER) was similar (data not shown). Briefly, the body weight changes of nondiabetic, diabetic sham control, moringa-diabetic, and metformin-diabetic groups were 8.3 ± 1.3, 15.3 ± 2.5, 12.3 ± 2.1, and 13.3 ± 3.4 g, respectively, and the FER was 4.8% ± 0.4%, 9.4% ± 1.8%, 8.1% ± 1.7%, and 7.9% ± 1.4%, respectively. These results indicate that M. oleifera inhibits weight gain and FER, which might be one of the reasons to ameliorate TG and LDL-C level.

Nephropathy is the major complication of diabetes and is the most common cause of end-stage renal disease.⁵⁰ Diabetic nephropathy is a complex disease involving many cell types and leading to structural and functional abnormalities, which include hyperfiltration with glomerular hypertension, renal hypertrophy, increased glomerular basement membrane thickness, tubular atrophy, and interstitial fibrosis. These events lead to the development of proteinuria, worsening systemic hypertension and ultimately progressive loss of kidney function.⁴² Similar results were obtained in rats by Adeymei and Elebiyo.⁵¹ We also conducted histopathology examinations of kidneys, and we found inflammation in blood vessels, increased thickness of Bowman's capsules and decreased glomerulus size. M. oleifera extract treatment improved kidney histopathology, showing reduced inflammation in the blood vessels, and the glomerulus size returned to a similar size as the normal group. In addition, we also investigated the expression of inflammation-related genes from kidneys. The treatment of kidney tissues with the M. oleifera significantly decreased the expression of inflammation-related genes including TNF-α, IL-1β, IL-6, COX-2, and iNOS.

We have demonstrated that M. oleifera leaf extract from Cambodia possessed potent antioxidant activities and decreased the blood glucose levels. Furthermore, the leaf extract protected the kidney against ROS-mediated damage by enhancing cellular antioxidant defenses and minimizing hyperglycemia in the M. oleifera extract group. Therefore, the M. oleifera leaf extract from Cambodia could be used as a source of natural antioxidants and antidiabetic agents and may have potential application as a therapeutic reagent.

Footnotes

Acknowledgment

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Agri-Bio Industry Technology Development Program (316027-5 and 116083-3) funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA).

Author Disclosure Statement

No competing financial interests exist.

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