Potential of Moringa oleifera to Improve Glucose Control for the Prevention of Diabetes and Related Metabolic Alterations: A Systematic Review of Animal and Human Studies

Abstract

Moringa oleifera (MO) is a multipurpose plant consumed as food and known for its medicinal uses, among others. Leaves, seeds and pods are the main parts used as food or food supplements. Nutritionally rich and with a high polyphenol content in the form of phenolic acids, flavonoids and glucosinolates, MO has been shown to exert numerous in vitro activities and in vivo effects, including hypoglycemic activity. A systematic search was carried out in the PubMed database and reference lists on the effects of MO on glucose metabolism. Thirty-three animal studies and eight human studies were included. Water and organic solvent extracts of leaves and, secondly, seeds, have been extensively assayed in animal models, showing the hypoglycemic effect, both under acute conditions and in long-term administrations and also prevention of other metabolic changes and complications associated to the hyperglycemic status. In humans, clinical trials are scarce, with variable designs and testing mainly dry leaf powder alone or mixed with other foods or MO aqueous preparations. Although the reported results are encouraging, especially those from postprandial studies, more human studies are certainly needed with more stringent inclusion criteria and a sufficient number of diabetic or prediabetic subjects. Moreover, trying to quantify the bioactive substances administered with the experimental material tested would facilitate comparison between studies.

Full text

nutrients
Review
Potential of Moringa oleifera to Improve Glucose
Control for the Prevention of Diabetes and Related
Metabolic Alterations: A Systematic Review of
Animal and Human Studies
Esther Nova 1, * , NoemíRedondo-Useros 1, Rosa M. Martínez-García2,
Sonia Gómez-Martínez 1, Ligia E. Díaz-Prieto 1and Ascensión Marcos 1
1
Immunonutrition Research Group, Department of Metabolism and Nutrition, Institute of Food Science and
Technology and Nutrition (ICTAN)—CSIC, C/Jose Antonio Novais 10, 28040 Madrid, Spain;
noemi_redondo@ictan.csic.es (N.R.-U.); sgomez@ictan.csic.es (S.G.-M.); ldiaz@ictan.csic.es (L.E.D.-P.);
amarcos@ictan.csic.es (A.M.)
2Department of Nursery, Physiotherapy and Occupational Therapy, Faculty of Nursery,
University of Castilla-La Mancha, 160071 Cuenca, Spain; rosamaria.martinez@uclm.es
*Correspondence: enova@ictan.csic.es; Tel.: +34-915-492-300 (ext. 231209)
Received: 2 June 2020; Accepted: 7 July 2020; Published: 10 July 2020


Abstract:
Moringa oleifera (MO) is a multipurpose plant consumed as food and known for its medicinal
uses, among others. Leaves, seeds and pods are the main parts used as food or food supplements.
Nutritionally rich and with a high polyphenol content in the form of phenolic acids, flavonoids and
glucosinolates, MO has been shown to exert numerous
in vitro
activities and
in vivo
effects, including
hypoglycemic activity. A systematic search was carried out in the PubMed database and reference
lists on the effects of MO on glucose metabolism. Thirty-three animal studies and eight human
studies were included. Water and organic solvent extracts of leaves and, secondly, seeds, have been
extensively assayed in animal models, showing the hypoglycemic effect, both under acute conditions
and in long-term administrations and also prevention of other metabolic changes and complications
associated to the hyperglycemic status. In humans, clinical trials are scarce, with variable designs
and testing mainly dry leaf powder alone or mixed with other foods or MO aqueous preparations.
Although the reported results are encouraging, especially those from postprandial studies, more
human studies are certainly needed with more stringent inclusion criteria and a sufficient number of
diabetic or prediabetic subjects. Moreover, trying to quantify the bioactive substances administered
with the experimental material tested would facilitate comparison between studies.
Keywords:
Moringa oleifera; diabetes mellitus; fasting glucose; glucose tolerance; antioxidant enzymes;
lipid metabolism; animal studies; human studies
1. Introduction
The Moringa oleifera (MO) tree, known as ‘drumstick tree’ belongs to the Moringaceae family. It is
the best known and most widely used of the thirteen species of the Moringa genus. It is originally
from the southern Himalayas to the north-east of India, Bangladesh, Afghanistan and Pakistan and is
nowadays cultivated in tropical and subtropical areas of Africa, America and Asia. It is a fast- growing
perennial tree that can measure up to 12 meters in height and displaying great ecological plasticity
since it is able to adapt to the most dissimilar conditions of the soil, temperature and precipitation,
being very resistant to the drought [
1
,
2
]. Its leaves are compound, and are arranged in groups of
leaflets with a length of 30–70 cm, the flowers have five unequal white petals and yellow stamens and
Nutrients 2020,12, 2050; doi:10.3390/nu12072050 www.mdpi.com/journal/nutrients

Nutrients 2020,12, 2050 2 of 28
the fruits in the form of pods contain 12 to 15 winged seeds per fruit and its cultivation is carried out
by sowing or using cuttings [3,4].
MO is considered a versatile plant due to its multiple uses. The leaves, green pods and seeds
are edible and are used as food as part of the traditional diet in many countries in the tropics and
subtropics. The green pods and leaves are consumed as vegetables and boiling is the most widely used
method of cooking; the seeds are ground to obtain a flour that is used together with the leaves in the
preparation of soups and with other flours (wheat or corn) to make bread and biscuits, improving their
nutritional quality [
5
,
6
]; the seeds can be consumed fresh or pounded, roasted or pressed into a sweet,
high quality oil [
7
]. The seed oil, due to its thermal and oxidative stability, is used for cooking and
as a solidifying agent in the production of margarine and other food products containing solid and
semi-solid fats, thus eliminating the process of hydrogenation [
8
,
9
]. Although its bitter and a little
astringent taste may be a barrier to acceptance, there is currently an increase in consumption of teas
prepared from MO leaves in western markets [10].
The flowers, pods, seeds and mainly the leaves are a source of essential nutrients and
nutraceuticals [
11
]. They contain protein, lipids (mainly omega-3 and omega-6 polyunsaturated
fatty acids, oleic acid and a low content of saturated fatty acids), carbohydrates, minerals (potassium,
calcium, magnesium and iron), vitamins (
β
-carotene,
α
-tocopherol and highly bioavailable folic acid)
and dietary fiber, and can be a food resource for people who are undernourished [
11
,
12
]. On a dry
matter basis, the total protein content measured in dry leaf powder ranges from 23 to 35%, which
is a high content compared to other local plants of common use [
13
,
14
]. Consistent with these data,
the analysis of 181 samples from different African and Asian countries showed a mean value of
30.3
±
4.9% [
15
]. Total dietary fiber was reported in the range of 20 to 28% by different authors [
16
–
18
].
The mean concentration of Ca, Cu, Fe, mg, and Zn in MO leaves collected from a garden in Jalisco
State of Mexico were 16,100, 9.6, 97.9, 2830 and 29.1 mg kg
−1
dry weight (dw), respectively [
13
] and the
overall mean concentrations of Ca, Cu, I, Fe, mg, Se and Zn in MO leaves from different localities in
Kenya were 18,300, 6.92, 0.218, 202, 5390, 4.25 and 35.6 mg kg
−1
dw, respectively [
19
]. The variability
is due to genetic background, soil, climate, season and plant age as well as processing and storage
procedures. Further, the use of different analytical techniques amplifies the variations [
18
]. Vitamin E
and
β
-carotene contents were 770 and 185 mg kg
−1
dw, respectively, in South African MO leaves [
16
].
Regarding fatty acid profile,
α
-linoleic acid (omega-3) showed the highest value (44.57%) in this
study and the total polyunsaturated fatty-acids (52.21%) were more abundant than saturated fatty
acids (43.31%) [16].
MO presents a wide variety of biological activities evidenced in
in vitro
experiments, showing
potent anti-oxidative, analgesic, cytoprotective, anti-ulcer, anti-hypertensive and immunmodulatory
actions as well as an inhibitory effect on proinflammatory mediators such as iNOS, COX-2, PGE-2,
TNF-
α
, IL-1
β
and IL-6 [
2
,
17
,
20
–
23
]. It is a popular medicine used in Asia and Africa for multiple
purposes, ascribed to the various parts of the plant, which, in addition, are used in many different
ways [
7
]. Its therapeutic value as a cardioprotective, hepatoprotective, neuroprotective, anti-asthmatic,
anti-tumor, antimicrobial, hypolipidemic, modulator of intestinal microbiota and anti-diabetic agent
derives from its phytochemical constituents such as alkaloids, phenolic compounds (coumarins, tannins,
flavonoids) and glycosides (saponins and glucosinolates) although the amount of these metabolites
varies according to the geographical location and the extraction method used [7,24–26].
The leaf is the most commonly used plant part for therapeutic purposes. The main phytochemical
compounds extracted from the leaves of MO include glucosinolates, flavonoids and phenolic acids
that have a protective effect against chronic diseases (arterial hypertension, diabetes mellitus, cancer,
metabolic syndrome and overall inflammation) [
27
]. Glucosinolates are highly represented in the
Brassicaceae family (cruciferous), belonging, as the Moringaceae family, to the order Brassicales.
They are known as mustard oils and are not bioactive until they are hydrolyzed (during the crushing
or breakage of the vegetable cells), by endogenous “myrosinase” enzymes resulting in the formation of

Nutrients 2020,12, 2050 3 of 28
thiocyanates, isothiocyanates and nitriles which constitute the active molecules with chemo-protective,
hypotensive and hypoglycemic effects [7,10,27,28].
On the other hand, the presence of phytates and oxalates, with mineral-binding activity that
decreases their absorption and saponins, which show favorable or detrimental nutritional and health
effects depending on the ingested amount [
29
], is to be considered when evaluating the safety of
long-term use and the overall guidelines of MO consumption as a functional food or nutraceutical.
Body fat accumulation, insulin resistance development and chronic inflammation are considered
the cornerstones of the metabolic alterations leading to diabetes mellitus (DM) type 2. The global
diabetes prevalence in 2019 was estimated to be 9.3% (463 million people), rising to 10.2% (578 million)
by 2030 and 10.9% (700 million) by 2045 [
30
]. Treating diabetic patients is one of the highest costs
of health care systems and it increases each year, which highlights the relevance of prevention and
specifically the level of information and awareness of the prediabetic patient. Five to 10% of prediabetic
patients will develop DM each year [
31
]; hence, identifying and treating prediabetes is relevant to
avoid or slow down DM establishment. Pharmacotherapy to control the progress of the disease is not
without negative side effects. More drawbacks of drug therapies are long-term loss of efficacy and
poor adherence to lifelong treatments [
32
]. On this basis, plants and herbs with hypoglycemic activity
could represent good alternatives, especially for those prediabetic patients who fail to make durable
lifestyle changes.
MO has a potent antioxidant activity, which can prevent and protect pancreatic cells from the
oxidative stress associated with the hyperglycemic state [
33
]. This capacity is attributed to the high
concentration of polyphenolic compounds such as flavonoids (myricetin, quercetin and kaempferol)
and phenolic acids (chlorogenic acid, caffeic acid and the most abundant, gallic acid). On the other
hand, the importance of isothiocyanates in glycemic control seems to be related to their ability to
reduce resistance to the action of insulin and hepatic gluconeogenesis [28].
The hypoglycemic effect of the MO leaves has also been associated with their fiber content and
the presence of flavonoids and phenolic acids through different mechanisms.
In vitro
experiments
have provided evidence that these molecules or the extracts rich in them inhibit the activity of
pancreatic
α
-amylase and intestinal
α
-glucosidase, decreasing intestinal absorption of glucose and
advanced glycation [
12
,
34
–
37
], thus reducing the risk of developing DM and improving glucose levels
in prediabetic and diabetic patients. There are other multiple potential mechanisms involved which
include e.g., the inhibition by quercetin glycosides of the Na
+
-dependent glucose uptake via the SGLT-1
transporter [
38
,
39
]. These mechanisms are numerous and will not be reviewed here; on the contrary,
the purpose of this work is to review the evidence of the potential use of MO for glucose control, both,
in animal models and human clinical trials.
2. Materials and Methods
We performed a systematic review of MO effects on the control of blood glucose levels according
to PRISMA statement guidelines.
The literature search was performed in PubMed database from August 2019 to April 2020 using
the terms “Moringa oleifera” and “diabetes” without year limits. Seventy-three papers were retrieved
(Figure 1). Articles were discarded if they were reviews (N =10) or reported only
in vitro
cell culture
experiments (N =9) or plant analysis studies (N =2). All papers covering animal models and human
studies were further considered. Among animal studies, six were excluded because they did not present
data on glucose control and involved pathologies basically independent from DM or its complications.
Among human studies, eight of them were excluded because they were related to plant uses among
populations, mainly determined through surveys. This left 38 articles, (35 on animals and three on
humans) which were included in Tables 1and 2, except for two animal studies, one of them considered
methodologically weak and another one testing a synthetic compound, which was not isolated from
M. oleifera. Regarding human studies, five additional studies not listed in PubMed were retrieved by
searching the reference lists in other included articles or in literature reviews.

Nutrients 2020,12, 2050 4 of 28
Table 1. Animal studies on the effect of M. oleifera on glucose control and biomarkers related to diabetes complications.
Model Treatment and Duration Measurements Evidences: MO Treated Animals vs. Untreated Ref
Studies with raw MO
Alloxan-induced diabetic
Sprague Dawley rats.
Normal Sprague
Dawley rats
MO dry leaf powder (50 mg/day,
gavage)
8 wk.
Body weight, BG, lipid profile.
Intestine histopathology, lactic acid
bacteria and Enterobacteriaceae (culture).
↓BG.
Prevented weight loss. No effect on lipid profile.
No histopathology observations.
No effect in BG in normal rats.
↓Enterobacteria enumeration
[40]
STZ-diabetic male
Wistar rats
Diets containing 2% and 4% MO
leaves or MO seeds
(±Acarbose, ACA)
2 wk.
BG (every 3 days), acetylcholinesterase
(AChE), butyrylcholinesterase (BChE)]
angiotensin-I converting enzyme (ACE),
arginase, CAT, GST and GSH-Px
activities, GSH and nitric oxide (NO)
levels in brain
↓BG (all treated groups). The highest reduction
occurred with 4% MO leaves +ACA.
↓AChE, BChE and ACE activities and ↑
antioxidant molecules (both preventive of
cognitive dysfunction)
[41]
Male spontaneously diabetic
Goto-Kakizaki rats
Normal male Wistar rats
MO leaf powder (200 mg/kg)
Single dose (glucose-MO solution,
oral admin.)
OGTT (BG at 10, 20, 30, 45, 60, 90 and 120
min and iAUC). Stomach, small intestine
and caecum content weights.
↓BG at 20, 30, 45, and 60 min. ↓iAUC.
↑stomach contents (⇒delayed gastric emptying).
↓BG at 10, 30 and 45 min in normal rats.
[39]
STZ-induced diabetic adult
male Wistar rats
MO seed powder (50 or 100 mg/kg)
in the diet
4 wk.
FBG, HbA1c, lipid peroxidation,
antioxidant enzymes, liver and renal
function, IgG, IgA, serum and kidney
IL-6 and kidney and
pancreas histopathology
Prevented weight loss, ↓FBG (35% and 45%, 50
and 100 dose, resp.) and ↓HbA1C (13% and 22%).
Improvement of all oxidative status parameters,
Igs and IL6; all approaching the negative control.
Restoration of the normal histology of both kidney
and pancreas. Both doses are effective but, overall,
the higher dose is more effective.
[42]
Studies with MO aqueous extract
STZ-diabetic female
Wistar rats
HFD-induced diabetic
C57BL/6 mice
Normal rats and mice
1) Aq MO, 100 mg/kg (in the diet) to
STZ-induced rats
2) Aq MO, 200 mg/kg (in the diet) to
HFD-fed mice.
Either, 2 single doses (2 days) or
3 wk.
FBG, OGTT, lipid profile, liver
marker enzymes.
↓
FBG,
↓
2 h glucose and AUC of glucose in OGTT.
↓SGOT and SGPT (HFD- and STZ-induced
animals), improved lipid profile (more significant
in HFD-mice than STZ-diabetic rats)
No differences in FBG and biochemical parameters
in normal rats and mice
[43]

Nutrients 2020,12, 2050 5 of 28
Table 1. Cont.
Model Treatment and Duration Measurements Evidences: MO Treated Animals vs. Untreated Ref
STZ-induced diabetic male
Wistar rats (sub, mild and
severely diabetic)
Normal Wistar rats
Aq MO (100, 200 and 300 mg/kg,
oral gavage)
Either, single dose or 21 days
BG and OGTT in response to single doses.
FBG, PPG, haemoglobin, total protein
and weight gain after 21 days.
BG and OGTT in response to single doses
↓
BG in OGTT (at 3 h post oral glucose): maximum
fall of 31.1% and 32.8% in sub-diabetic and
mild-diabetic rats, respectively, occurred always
with the 200 mg/kg dose.
↓FBG (69.2%) and ↓PPG (51.2%) in severely
diabetic rats on a 21-day treatment.
↑
Hemoglobin
(10.9%) and total protein (11.3%)
↓BG in normal rats: maximum fall of 26.7%
occurred 6 h after 200 mg/kg, single dose.
↓BG in OGTT (at 3 h post oral glucose) in normal
rats: maximum fall of 29.9% with 200 mg/kg dose
[44]
Adult male Wistar rats
MO tea (10, 20 and
30 mL/kg, gavage)
Single dose
OGTT (4 g/kg b.w. of glucose, 30 min
after MO tea)
↓16, 18 and 6% total PPG ⇒Lower doses are
more efficient. [45]
Alloxan-induced diabetic
Wistar rats.
Normal Wistar rats
Aq MO (250 mg/kg, oral admin.)
18 days
BG, hepatic lipid peroxidation and
antioxidant enzyme activities,
histoarchitecture of hepatic and
pancreatic tissues, gene expression of
glycogen synthase (GS), pyruvate
carboxylase (PC) and caspase 3, and SOD
and CAT activities.
↓BG, prevented organ changes and significantly
restored all measures. Normalized the expression
of apoptotic, gluconeogenic, and glycogenic genes
in hepatic tissue
No effect on BG in normal rats. ↑liver GSH and
GS expression. No other significant changes in
normal rats.
[46]
Alloxan-induced diabetic
female Wistar rats.
Normal Wistar rats
Aq MO (250 mg/kg, oral admin.)
18 days
Body weight, BG, lipid profile, lipid
peroxidation, histoarchitecture of hepatic
and pancreatic tissues, expression of
pyruvate kinase (PK), pyruvate
carboxylase (PC), and fatty acid synthase
(FAS) in liver.
↓BG
↑Body weight, ↓TG and MDA. Normalized the
expression of enzymes of gluconeogenesis and
fatty acid synthesis. Normalized histological
structures.
No effects in normal rats.
[47]
Alloxan-induced diabetic
Wistar rats
MO extracts: Aq, Me MO and Et
MO (50% water: 50% alcohol and
100% alcohol, dose range:
200–400 mg/kg, oral admin.)
24 days
FBG
All extracts and doses ↓FBG around 70–87%. Aq
MO (300 mg/kg) reduction was 82%. All extracts
showed body weight restoration capacity.
[48]

Nutrients 2020,12, 2050 6 of 28
Table 1. Cont.
Model Treatment and Duration Measurements Evidences: MO Treated Animals vs. Untreated Ref
Alloxan-Induced diabetic
albino mice
Normal albino mice
Aq MO (100 mg/kg, oral gavage)
14 days
FBG, insulin, HOMA-IR, TAC, creatinine,
blood urea nitrogen (BUN). Percentage
CD44, CD69 and IFN-γpositive cells
in PBMCs
↓
FBG,
↑
(NS) insulin,
↓
HOMA-IR.
↓
creatinine and
BUN and ↑TAC and IFN-γ.
↑Insulin in normal rats and no effect on FBG and
HOMA-IR. No effect in other
parameters measured.
[49]
STZ-induced diabetic male
Sprague-Dawley rats
Normal Sprague-Dawley rats
Aq MO (200 mg/kg, oral gavage)
8 wk
FPG; GSH, lipid peroxidation,
histopathology and morphometric
analyses of pancreas
↓FPG (62%)
↓MDA and ↑GSH, normalization of
histopathological and morphometric changes
No effects in normal rats.
[50]
STZ-induced diabetic rats Aq MO (100 mg/kg, oral gavage)
24 wk.
Body weight, BG and HbA1C.
TNF-α, IL-1β, VEGF, PKC-β, GSH, SOD,
CAT in retinae. Retinal leakage and
retinal vessel caliber (arteriolar and
venular) and basement
membrane thickness.
↓BG (33%), and HbA1C (40%).
Preserved weight gain.
↑Antioxidant parameters, ↓inflammatory and
angiogenic parameters, ↓all morphological and
structural alterations of the retinae.
[51]
VHFD-induced obese male
C57BL/6J mice
Aq MO (5% MO in VHFD,
66 mg/d MIC)
12 wk.
OGTT at 4th, 8th and 12th wk., plasma
insulin, leptin, resistin, IL-1βand TNFα,
total cholesterol and triglycerides. Liver
histology and gene expression: TNF-α,
IL-1β, IL-6, G6Pase, PEPCK and GcK.
Insulin signaling proteins (liver and
muscle) and lipolysis-related gene
expression and protein levels (adipose
tissue and liver).
↓
BG and
↓
AUC of glucose at 8th and 12th wk (NS
↓at wk. 4th).
↓Weight gain, ↓body fat accumulation, ↓plasma
insulin, leptin, resistin, cytokines and cholesterol,
↓Hepatic G6Pase and TNF-αexpression,
improved insulin signaling (
↑
IRSs, protein kinases,
PI3K, and GLUT4) and liver lipolytic
protein levels.
[28]
High fructose diet-induced
diabetic male Sprague
Dawley rats
Aq MO (300 mg/kg, oral admin.)
4 wk.
FSG, Insulin, HOMA-IR, testosterone and
FSH; MDA, SOD, CAT in liver; insulin
receptor (IR), IRS-1, GLUT-4 & GLUT-5
and SOD, steroidogenic acute regulatory
protein (StAR) and 3β-hydroxysteroid
dehydrogenase (3β-HSD) expression
in liver
No effect on FSG; ↑insulin and ↓HOMA-IR,
↓MDA, ↑SOD, CAT and testosterone.
Improvement of the down-regulation of the
insulin signaling pathway. Improved regulatory
proteins of testicular function
[52]

Nutrients 2020,12, 2050 7 of 28
Table 1. Cont.
Model Treatment and Duration Measurements Evidences: MO Treated Animals vs. Untreated Ref
Alloxan-Induced diabetic
Unib:SW (Swiss) mice
Protein isolate of MO leaves
(500 mg/kg) (i.p. and oral admin.)
Single dose and 7 days
BG (5 h after single dose and 4 h after
daily treatment at 3rd and 7th day),
insulin (5 h after single dose), liver MAD,
CAT and SOD.
i.p. admin.: ↓BG, ↓liver MDA and ↑CAT. Did not
change serum insulin.
Oral admin.: No effect on BG (due to
protein digestion).
[53]
Studies with MO methanolic or ethanolic extracts
STZ-induced diabetic Long
Evan rats
Et MO (0.5 g/kg, oral admin.) and
Et MO with glucose (OGTT).
Single dose, both
FBG, OGTT (2.5 g/kg oral glucose),
insulin, intestinal glucose absorption by
perfusion technique through the pylorus.
↓
FBG at 90 min post Et MO and
↓
BG in OGTT. No
change in insulin.
↓Glucose absorption.
[36]
HFD-induced obese male
C57BL/6J mice
Me MO (250 mg/kg, oral admin.),
fermented (FM) and non-fermented
(NFM). Fermentation starter: 3 LAB
strains isolated from
cabbage kimchi
10 wk.
Glucose tolerance (2 g/kg glucose, ip.
injection) at 8th wk., hepatic lipid
accumulation, expression of proteins and
genes involved in glucose and
lipid regulation
FM: ↓AUC glucose, ↓hepatic lipid accumulation.
Upregulation of genes related to lipid metabolism.
↓Oxidative stress and lipotoxicity in muscle. ↓
proinflammatory cytokine expression in muscle,
and liver tissues.
NFM: No effect on glycemic response. ↓Hepatic
lipid accumulation. Mixed effects on
proinflammatory cytokine expression and levels in
different tissues.
[54]
Alloxan-induced
diabetic rats
Me MO (300 or 600 mg/kg,
oral gavage)
6 wk.
Food intake, body weight, intraperitoneal
glucose tolerance (IPGT, 30, 60 and 120 min
post 2 g/kg glucose administration),
serum glucose, insulin, and lipids, liver
and muscle glycogen synthase activity,
glycogen content, and glucose uptake.
Prevented weight loss, ↓BG and ↑Insulin, ↓BG at
all-time points in IPGT.
Improved lipid profile, increased glycogen
synthase activity and glycogen content in muscle
and liver and improved glucose uptake.
[55]
Obese C57Bl/6J male mice
fed VHFD
Normal C57Bl/6J male mice
fed LFD
Et MO (seed, 47% MIC) in the diet.
Average dose: 161 ±19 mg
MIC-1/kg in VHFD; 335 ±23 mg
MIC-1/kg in LFD.
12 wk.
Body weight, body composition, OGTT
at week 2nd, 4th, 6th, 9th and 12th; liver
lipids, IL-1β, IL-6, TNF-α, iNOS, NQO-1
gene expression, intestinal microbiota
composition and load.
↓AUC glucose in the VHFD animals.
Similar AUC glucose in LFD animals (except at
week 9, treated <untreated).
↓body weight, ↓adiposity; ↓iNOS expression, ↑
antioxidant NQO-1 expression (both diets); ↓
bacterial load, modulation of bacterial community
(both diets)
[56]
STZ/HFD-induced diabetic
male Wistar rats
Normal Wistar rats
Me MO (250 mg/kg, gavage)
6 wk
FPG, kidney lipid peroxidation, CAT,
GPx, SOD activities, GSH and
inflammatory biomarkers.
↓
FPG,
↓
kidney weight and relative kidney weight.
↓Kidney MDA, ↑CAT (and ↑NS SOD), ↓
pathological observation in histology.
↓FPG, ↑SOD, ↓GPx (and ↑NS CAT) in
normal rats.
[57]

Nutrients 2020,12, 2050 8 of 28
Table 1. Cont.
Model Treatment and Duration Measurements Evidences: MO Treated Animals vs. Untreated Ref
STZ-induced diabetic
female Wistar rats
Normal Wistar rats
Me MO (250 mg/kg, gavage)
6 wk.
FSG, liver weight and enzymes, lipid
profile, liver antioxidant capacity,
inflammatory cytokine levels
and histopathology.
↓FSG
↓
Liver weight, SGOT, ALP, LDL and cholesterol.
↓
IL-6, TNF-α, and MCP-1. Improvement of liver
histological alterations
Non-significant ↓FSG in normal rats.↓Liver
enzymes and ↑HDL in normal rats.
[58]
STZ-induced diabetic male
Sprague Dawley rats.
Normal male Sprague
Dawley rats.
Me MO (300 mg/kg, oral gavage)
60 days
FBG (glucometer), FSG (ion exchange),
HbA1C, Insulin, SOD, CAT, GPx, GR,
GSH, TBARS.
↓FBG, FSG, and HbA1c and ↑increased insulin.
↑Activities of antioxidant enzymes in the heart of
diabetic rats.
No effects in normal rats.
[59]
STZ-induced diabetic
Wistar rats
Normal Wistar rats
Me MO (pods, 150 and 300 mg/kg,
oral admin.)
3 wk.
BG, insulin, total protein and albumin
and NO. Pancreatic lipid peroxidation,
SOD, GSH, CAT and glycogen
and histopathology.
↓BG, ↑insulin, total protein, albumin and NO. ↓
MDA and ↑antioxidant activities in pancreas and
reversed the histoarchitectural damage.
No changes in normal rats.
[60]
STZ-diabetic Wistar rats Me MO (200 mg/kg, gavage)
3 wk.
FBG, weight, supercomplex formation,
ATPase activity, ROS production, GSH
and GR levels, lipid peroxidation and
protein carbonylation of
liver mitochondria
↓FBG: 86 ±4.2 mg/dL vs. 229 ±9.05 mg/dL.
↑GSH and GR; ↓lipid peroxidation and protein
carbonylation of liver mitochondria.
[61]
db/db mice Et MO (150 mg/kg)
5 wk.
FPG, lipid profile, kidney histology and
expression of inflammation markers.
↓FPG (36%), TG and LDL. ↑insulin.
↓kidney histopathological damage. ↓expression
of kidney inflammatory markers
[62]
Alloxan-Induced diabetic
Wistar rats
Et MO (200 mg/kg, twice daily,
oral admin.)
5–6 days
FBG, electrolytes: potassium (K), sodium
(Na), chloride (Cl−), bicarbonate and
lactate dehydrogenase (LDH) enzyme.
↓FBG.
No effect on weight. No cytotoxicity. ↓
Bicarbonate, ↑anion gap value (⇒acidosis, but
lower that metformin).
[63]
Alloxan-induced diabetic
Charles Foster strain male
albino rats
Et MO (stem bark, 250 mg/kg,
oral admin.)
1 wk.
FBG and urine sugar ↓FBG and nil urine sugar detected. [64]
Alloxan-Induced diabetic
rats with/w-out sitagliptin
treatment
Et MO (300 mg/kg, oral gavage)
42 days
FBG and other glycaemic control
parameters, insulin, body weight, retinal
microvasculature on lenticular
opacity/morphology
No difference in FBG between day 42nd and day
1st. ↓random BG (42nd vs. 1st day). Overall, less
anti-hyperglicemic effect than sitagliptin alone. No
changes in insulin secretion and body weight.
No prevention or amelioration of retina lesions.
[65]

Nutrients 2020,12, 2050 9 of 28
Table 1. Cont.
Model Treatment and Duration Measurements Evidences: MO Treated Animals vs. Untreated Ref
Alloxan-induced
Swiss-Webster male mice
A) N-Hexane extract of MO seeds
(40, 60 and 80 mg/kg, i.p.)
B) β-sitosterol (BSL) fraction from
hexane extract (18, 25 and
35 mg/kg, i.p.)
Single dose (acute) and 8 days
BG (acute: 1, 2 and 6h; subchronic: 1st,
3rd, 5th, and 8th day, 6 h post daily
injection); Insulin, HbA1C, CAT, lipid
peroxidation (8 wk post-treatment end);
Diabetic painful neuropathy measures
(hot plate latency, tail flick latency and
von Frey filaments test, 8 wk
post-treatment end)
Acute: ↓BG at 2 h and 6 h with all doses of MO
and BSL.
Subchronic: ↓BG from day 1 to day 8 with MO
and BSL (maximum ~ 70% reduction on day 8th).
↑Insulin several fold, ↓HbA1C, improved
antioxidant markers, 8 wk. post-treatment end. ↑
body weight (both).
Significantly improved thermal hyperalgesia and
tactile allodynia (MO more powerful than BSL).
[66]
db/db mice
Niazirin (10 mg/kg and 20 mg/kg,
extracted and concentrated from
seeds. 95% purity. Oral gavage)
4 wk.
FBG and Insulin, HOMA-IR, OGTT.
Plasma TNF-alpha, IL-10, LDL, HDL, TC,
TG, NEFA levels; Liver glycogen, HK, PK,
G6Pase and PEPCK activities; Liver
histological analysis; Liver AMPK,
p-AMPK, FAS, p-ACC, SREBP-1, PPAR-
α
,
SirT1, FOXO1, HNF-4
α
, PGC-1
α
, PFKFB-3.
↓FBG and insulin with improved HOMA-IR
(both doses).
OGTT: lower BG at 90 and 120 min (both doses)
Improved glucose uptake and glycogen storage in
the liver through AMPK pathway.
Improved lipid profile; reduced FA synthesis and
induced FA oxidation through AMPK pathway.
[67]
STZ-induced diabetic
ICR mice
4 compounds isolated from MO
seeds by macroporous resin
adsorption and chromatography
(20 mg/kg, i.v.)
2 wk.
BG
↓BG, 3 compounds: 1)
N,N0-bis{4-[(α-l-rhamnosyloxy)benzyl]}thiourea,
2) niazirin A, 3)
S-Methyl-N-{4-[(α-l-rhamnosyloxy)benzyl]}
thiocar bamate
No effect:
4-[(6-deoxy-
α
-l-mannopyranosyl)oxy]-benzaldehyde
[68]
Extracts are from leaves unless otherwise specified; MO: M. oleifera; Et MO: ethanolic extract of MO; Aq MO: aqueous extract of MO; Me MO: methanolic extract of MO. BG: Blood glucose;
STZ: Streptozotocin, OGTT: Oral glucose tolerance test; iAUC: Incremental area under the curve; CAT: catalase, GST: Glutathione S-transferase; GSH-Px: Glutathione peroxidase; GSH:
Reduced glutathione; FBG: Fasting blood glucose FBG; HbA1C: glycated hemoglobin; Igs: Immunoglobulins; HFD: high-fat diet; SGOT: Serum Glutamic Oxaloacetic Transaminase;
SGPT: Serum Glutamate Pyruvate Transaminase; PPG: Postprandial glucose; SOD: Superoxide dismutase; MDA: Malondialdehyde; HOMA-IR: Homeostatic model assessment for
insulin resistance; TAC: Total antioxidant capacity; FPG: Fasting plasma glucose; VEGF: vascular endothelial growth factor; PKC: protein kinase C; FSG: Fasting serum glucose; VHFD:
Very high-fat diet; MIC: Moringa isothiocyanates; G6Pase: Glucose-6-phosphatase; PEPCK: phosphoenolpyruvate carboxykinase; GcK: Glucokinase; AUC: Area under the curve; NS:
Non-significant; IRSs: Insulin receptor substrates; PI3K: fosfatidilinositol- 3-kinasa; GLUT-4 and -5: Glucose transporter 4 and 5; i.p.: intraperitoneal; LFD: Low-fat diet; TNF-
α
: Tumor
necrosis factor
α
; iNOS: Inducible nitric oxide synthase; NQO-1: NAD(P)H dehydrogenase [quinone]-1; FSG: Fasting serum glucose; ALP: Alkaline phosphatase; LDL: Low density
lipoprotein; MCP-1: Monocyte chemotactic protein 1; GR: Glutathion reductase; NO: Nitric oxide; TG: Triacylglycerides; HDL: High density lipoprotein cholesterol; NEFA: Non-esterified
fatty acids; HK: Hexokinase; PK: Piruvate kinase; AMPK: 5
0
-AMP activated protein kinase; p-AMPK: Phosphorylated AMPK, FAS: Fatty acid synthase; pACC: Phospho acetyl-CoA
carboxylase; SREBP-1: Sterol regulatory element-binding protein; PPAR-
α
: peroxisome proliferator-activated receptor; SirT1: Sirtuin 1; FOXO1: Forkhead box protein O1; HNF-4
α
:
Hepatocyte nuclear factor 4 alpha; PGC-1
α
: Peroxisome proliferator activated receptor-1
α
; PFKFB-3: Phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3; i.v.: Intravenous.
↑
:Higher in
MO treated animals than control,↓: Lower in MO treated animals than control.

Nutrients 2020,12, 2050 10 of 28
Table 2. Evidence from human studies on the effects of M. oleifera on glycemic control and related parameters in healthy and type 2 diabetic adults.
Treatment and
Duration Study Design Subjects Measurements Results
Compared to Baseline
Results
Compared to Control Group Ref.
Meal containing MO
leaf powder (20 g)
C: Control meal.
2 single occasions
Randomized,
placebo controlled,
crossover, PP
10 healthy adults
(6 W/4M)Age: 42
±
11 y
BMI: No data
17 type 2
diabetic patients
(9 W/8 M)
Age: 62 ±9 y
BMI: 25.2 ±4.3 kg/m2
Fasting glucose
(finger prick and
glucometer) and
postprandial glucose at
30, 60, 90, 120, 150, and
180 min from the
beginning of the meal
–
Healthy: ↔glycemic response
Diab: ↓blood glucose
from 60 to 180 min. ↓
increment form baseline at 90,
120 and 150 min.
[
12
]
Cookies containing MO
leaf powder (5% w/w)
C: Control cookies
Isocaloric and
containing 50 g
available carbohydrates
2 single occasions
Randomized
single-blinded,
placebo controlled
crossover, PP
20 Healthy subjects
(10 W/10 M)
Age: 24.1 ±1.33 y
BMI: 22.0 ±3.88 kg/m2
Fasting and postprandial
blood glucose (finger
prick and glucometer)
at 15, 30, 45, 60, 90 and
120 min.
Appetite, hunger and
palatability scales
–
↓non-significantly iAUC of
glucose (P =0.077).
↓blood glucose at 30 and
45 min
↓Hunger ratings
[
69
]
MO leaf capsules (4 g/d)
C: Placebo capsules.
4 weeks
Randomized,
placebo controlled,
parallel
32 Therapy-naïve
type 2 diabetics
(15 W/17 M)
Age: 50–60 y
BMI: 27.5 kg/m2
9-point blood glucose
(finger prick and
glucometer) along 3
consecutive days.FPG
and HBA1C
levels.Creatinine and
liver enzymes (ALT, AST)
↔Fasting plasma
glucose and HbA1C
↔Creatinine, ALT, AST.
↔Fasting plasma glucose and
HbA1C
↔mean daily BG, mean
premeal, and mean postmeal
BG.
↔Creatinine, ALT and AST
[
70
]
MO leaf powder (7 g/d)
in recipes in daily diet
C: No supplementation
3 months
Randomized,
controlled, parallel
60 Healthy
postmenopausal women
(60 W/0 M)
Age: 45–55 y
BMI: No data
FBG, hemoglobin,
ascorbic acid, retinol,
glutathione peroxidase,
superoxide dismutase
and malondialdehyde
↓FBG↑Blood
haemoglobin
↑Ascorbic acid, retinol,
glutathione peroxidase
and superoxide
dismutase
↓Malondialdehyde
↓FBG, ↑Blood haemoglobin
↑
Ascorbic acid and superoxide
dismutase
↓Malondialdehyde
[
71
]

Nutrients 2020,12, 2050 11 of 28
Table 2. Cont.
Treatment and
Duration Study Design Subjects Measurements Results
Compared to Baseline
Results
Compared to Control Group Ref.
MO leaf tea (200 mL or
400 mL)
C: Distilled water
Single occasions
Randomized,
controlled,
parallel, PP
15 Healthy subjects
(0 W/15 M)
Age: 20–29 y
BMI:21.6 kg/m2
In 3 groups, N =5
OGTT (50 g glucose) 30
min after MO tea oral
dose (finger prick and
glucometer)
–
↓glycemia (17% [200 mL] and
19% [400 mL] reduction).
Higher reduction at 30 min
with lowest amount (22.8 vs.
17.9%)
[
45
]
MO leaf powder (8 g/d)
C: No supplementation
40 days
Randomized,
controlled
22 type 2 diabetics
(8W/14 M)
Age: 40–60 y
BMI:18.5–35 kg/m2
FBG, PPG, lipid profile
(methods unspecified)
↓FBG and PPG
↓Total cholesterol, LDL
and TAG
No statistical test performed
[
72
]
MO leaf tablet
(2 units/day)
C: No supplementation
90 days
Intervention
controlled
60 type 2 diabetics on
sulfonylurea
medication
(gender unspecified)
Age: 40–58 y
BMI: 20–25 kg/m2
HbA1c and PPG two
hours after a meal
↓HbA1
(7.4% reduction)
↓PPG
↔HbA1 and PPG
[
73
]
MO leaf
powder capsules.
Dosages: 0, 1, 2 and 4 g.
C: 4 empty capsules.
4 single days separated
by 2 wk.
Oral single
dose study
10 Healthy volunteers
(5W/5M)
Age: 29 ±5 y
BMI: 18.5–23 kg/m2
Plasma glucose
and insulin
at intervals during 6 h
after single dose of MO.
Blood urea nitrogen,
creatinine, AST and ALT
at the first and forth visit
–
4 g: ↑plasma insulin
↑insulin AUC and
↑74% AUC of
insulin/glucose ratio
↔Plasma glucose, blood urea
nitrogen, creatinine, AST
and ALT
[
74
]
–: No applicable;
↔
: No changes;
↓
: Decreased;
↑
: Increased; ALT: Alanine transaminase; AST: Aspartate transaminase; BG: Blood glucose; BMI: Body Mass Index; FBG: Fasting blood
glucose; FPG: Fasting plasma glucose; HbA1c: Glycated haemoglobin; iAUC: Incremental area under the curve; LDL: Low-density lipoprotein; M: Men; MO: M. oleifera; PPG: Postprandial
glucose; TAG: Triacylglicerides; W: Women.

Nutrients 2020,12, 2050 12 of 28
Nutrients 2020, 12, x FOR PEER REVIEW 4 of 29
Figure 1. Flow chart of the selection of the animal and human studies included. DM: diabetes mellitus.
All animal studies with glucose control related measurements, both in diabetes models and in
normal healthy animals are presented in Table 1, which includes also a summary of other evidences
related to DM-associated pathophysiological complications. Human studies are presented in Table 2.
Finally, the ample set of measurements, from organ to molecular measurements, are systematically
classified and further detailed in Table 3 and Table 4.
3. Scientific Evidences of M. oleifera’s Effect on Glucose Control in Animal Models
Many studies have investigated the hypoglycemic effect of different parts of MO in animal
models, mainly the aqueous or methanolic extracts of leaves and, secondly, seeds. Dry leaves have
also been employed as powder. These studies are usually performed in streptozotocin (STZ) or
alloxan-induced diabetic rats and a fewer other studies have been performed in obese animals fed
high-fat diets (HFD). The three of them are used as models of DM. The first two models rely on
chemical destruction of pancreatic β-cells. Both chemicals are employed as cytotoxic glucose
analogues that tend to accumulate in pancreatic β cells through glucose transporter 2 (GLUT2).
However, depending on the dose of chemical employed, usually through intraperitoneal injection,
more resemblance to Type-1 or Type-2 diabetes is observed [40,41]. Moreover, there is some
controversy as to whether alloxan and STZ can be used to create type-2 diabetes models since both
exert toxicity on β-cells (alloxan through reactive oxygen species [ROS] formation and STZ through
DNA methylation that causes a chain of damages leading to DNA fragmentation) instead of inducing
insulin resistance, which is the main characteristic in type-2 diabetes. Only at low dose and in
combination with HFD [42] or when used with neonatal rats that will develop hyperglycemia in the
adult age [36], can STZ be considered to induce type-2 diabetes [40,41]. This is an important point
that has not been consistently approached in the experiments performed and the corresponding
published works.
In animal models of diabetes, different approaches have been used to administer MO to the
animals or incorporate it to the diet. Mainly two ways have been used, either delivery of any type of
previously prepared extract in aqueous solution through oral gavage or incorporation of dry material
(either lyophilized extracts or blended dry leaves) to the normal diet. As an example, Khan et al. [43]
prepared an aqueous extract, obtained through maceration of leaf powder during approximately 24
h, with continuous stirring and then filtration and lyophilization to get a solid residue that is later
incorporated to the standard diet.
Figure 1.
Flow chart of the selection of the animal and human studies included. DM: diabetes mellitus.
All animal studies with glucose control related measurements, both in diabetes models and in
normal healthy animals are presented in Table 1, which includes also a summary of other evidences
related to DM-associated pathophysiological complications. Human studies are presented in Table 2.
Finally, the ample set of measurements, from organ to molecular measurements, are systematically
classified and further detailed in Tables 3and 4.
Table 3. Effects of M. oleifera on antioxidant capacity and inflammation protection in diabetic animal models.
MO Material Animal Model Organ or
Biological Sample
Evidences
MO Treated vs. Untreated Ref.
Gene expression of inflammation markers
Me MO (FM)
ME MO (NFM) Obese (HFD) Liver and muscle ↓IL-6 and TNF-α.↓IL-1β, only muscle
↓IL-6. ↓(NS) TNF-α[54]
Et MO Diabetic Kidney ↓TNF-α, IL-1β, IL-6, COX-2 and iNOS [62]
Et MO (seed) Obese (VHFD)
Normal (LFD) Liver and intestine
↓iNOS in intestine of VHFD and LFD.
↓iNOS in liver of VHFD. No effect on
IL-1β, IL-6 and TNF-α.
↑NQO-1
[56]
Aq MO Diabetic skin wound tissues ↓TNF-α, IL-1β, IL-6, COX-2 and iNOS
↑VEGF. [75]
Aq MO Obese (VHFD) Ileum and liver
Adipose tissue
↓TNF-α.↓(NS) IL-6 and IL-1β.
↑Adiponectin [28]
Inflammatory cytokines
Me MO Diabetic Liver ↓TNF- α, IL-6 and MCP-1 [58]
Aq MO Obese (VHFD) Plasma ↓TNF-αand IL-1β[28]
MOP (seeds) Diabetic Blood and kidney ↓IL-6 [42]
Aq MO Diabetic and
normal PBMC ↑IFN-γ[49]
Niazirin (seed) Diabetic Plasma ↓TNF- α
↑IL-10 [67]
Oxidative status
Me MO Diabetic
Normal Liver ↑(NS) ORAC
↑ORAC [58]
Me MO Diabetic Kidney ↓MDA [57]

Nutrients 2020,12, 2050 13 of 28
Table 3. Cont.
MO Material Animal Model Organ or
Biological Sample
Evidences
MO Treated vs. Untreated Ref.
Me MO (pods) Diabetic Pancreas ↓MDA [60]
Me MO Diabetic Liver
(mitochondria) ↓MDA and ↓protein carbonilation [61]
Aq MO Diabetic Serum ↓MDA [47]
Aq MO Diabetic Liver ↓MDA [46]
MOP (seed) Diabetic Serum and kidney ↓MDA [42]
MO leaf/MO
seed Diabetic Brain ↓MDA [41]
Aq MO Diabetic Brain, liver, kidney,
pancreas and spleen
↓MDA [76]
Aq MO Diabetic Pancreas ↓MDA [50]
Aq MO Diabetic liver ↓MDA [52]
MO (protein
isolate) Diabetic Liver ↓MDA [53]
Me MO Diabetic Heart ↓MDA, HP and CD [59]
Antioxidant enzyme activity
Me MO Diabetic and
Normal Kidney
↑CAT (NS in normal)
↑SOD (NS in diabetic)
↓G-Px (NS in diabetic). No effect on GST
[57]
Me MO (pods) Diabetic Pancreas ↑GSH, SOD and CAT [60]
Aq MO Diabetic Pancreas ↑GSH [50]
Aq MO Diabetic liver ↑SOD, CAT [52]
Aq MO Diabetic Liver ↑SOD, CAT, GSH [46]
Aq MO Diabetic Plasma ↑TAC [49]
MOP (seed) Diabetic Serum and kidney ↑CAT, SOD and GSH [42]
MO leaf/MO
seed Diabetic Brain ↑CAT, G-Px, GST, GSH [41]
Aq MO Diabetic
Brain, liver, kidney,
pancreas and spleen.
Liver and pancreas
↑CAT, SOD
↑GST [76]
MO (protein
isolate) Diabetic Liver ↑CAT
No effect on SOD [53]
Me MO Diabetic Heart ↑CAT, SOD, G-Px, GR and GSH [59]
Me MO Diabetic Liver
(mitochondria) ↑GSH and GR [61]
Cholinergic dysfunction (associated to cognitive impairment as in diabetes encephalopathy)
MO leaf/MO
seed Diabetic Brain ↓AChE, BChE and ACE [41]
Extracts are from leaves unless specified in brackets; MO: M. oleifera; Me MO: Methanolic extract of MO; FM:
Fermented MO leaves; NFM: Non-fermented MO leaves; Et MO: Ethanolic extract of MO; Aq MO: Aqueous extract
of MO; MICs: MIC-1 (4-[(
α
-Lrhamnosyloxy)benzyl]isothiocyanate) and MIC-4 (4-[(4-O-acetyl-
α
-Lrhamnosyloxy)
benzyl]isothiocyanate); MOP: MO powder; HFD: High-fat diet; VHFD: Very-high-fat diet; NS: Non-significant
trend; IL-6: Interleukin-6; TNF-
α
: Tumor necrosis factor-
α
; IL-1
β
: Interleukin-1
β
; COX-1: Cyclooxygenase-2;
iNOS: inducible nitric oxide synthase; NQO-1: NAD(P)H dehydrogenase [quinone]-1; TAC: Total antioxidant
capacity; IFN-
γ
: interferon-
γ
; VEGF: vascular endothelial growth factor; MCP-1: monocyte chemotactic protein;
IL-10: Interleukin-10; ORAC: Oxygen radical absorbance capacity; MDA: Malondialdehyde; TBARS: Thiobarbituric
acid-reactive substances; HP: Hydroperoxides; CD: Conjugated dienes; CAT: Catalase; SOD: Superoxide dismutase;
GSH: Glutathione; G-Px: Glutathione peroxidase; GST: Glutathione-S-transferase; GR: Glutathione reductase; AChE:
Acetylcholinesterase, BChE: Butyrylcholinesterase; ACE: Angiotensin-I converting enzyme. ↑: Higher; ↓: Lower.

Nutrients 2020,12, 2050 14 of 28
Table 4.
Effects of M. oleifera on lipids, histopathology and gene expression in diabetic animal models.
MO Material Animal Model
Organ or
Biological
Sample
Evidences
MO Treated vs. Untreated Ref.
Accumulation of lipids in tissues
Me MO (FM
and NFM) Obese (HFD) Liver ↓Hepatic adiposity (H&E staining) [54]
Et MO (seed) Obese (VHFD) Liver ↓Liver lipids (Folch’s method
with modifications) [56]
Aq MO Obese (VHFD) Liver ↓Liver lipids (H&E staining) [28]
Niazirin (seed) Diabetic Liver ↓Hepatic lipid accumulation
(H&E staining) [67]
Circulating lipids
Aq MO Obese (VHFD) Plasma ↓Cholesterol [28]
Et MO Diabetic Plasma ↓TG and LDL-C [62]
Aq MO Diabetic Serum ↓TG. No effect on TC [47]
Me MO Diabetic Serum ↓TG, total and LDL-C and ↑HDL-C [55]
Niazirin (seed) Diabetic Plasma ↓LDL-C, TG and NEFA and ↑HDL-C.
↓TC (high dose only). [67]
Aq MO
Diabetic (2
models: HFD
and STZ)
Serum
↓
TC, TG, VLDL-C, and LDL-C.
↑
HDL-C
Smaller level of restoration of lipid profile
in STZ- diabetic than in HFD-diabetic
[43]
Histopathology and organ functionality
Et MO Diabetic Kidney Restored histopathological damage in
renal tissue [62]
Aq MO Diabetic Serum ↓GOT and GPT enzyme. [43]
Aq MO Diabetic Pancreas and
liver Prevented histoarchitectural changes [46,
47]
MOP (seed) Diabetic Kidney and
pancreas
Restored the normal histology of kidney
and pancreas [42]
Niazirin (seed) Diabetic Liver Restored NAFLD score and hepatocyte
structure. [67]
Me MO Diabetic Heart Improved histopathology [59]
Me MO Diabetic Liver Improved histopathology, ↓GOT and
ALP [58]
Me MO Diabetic Kidney Improved histopathology [57]
Me MO (pods) Diabetic Pancreas MO reversed the histoarchitectural
damage of islets cells [66]
Gene expression of lipid metabolism and glucose metabolism
Aq MO Obese (VHFD) Liver ↓Lipogenic proteins (FAS, SREBP1 and
FSP27) and ↑lipolytic ATGL. ↓G6Pase [28]
Fermented MO
leaf Obese (HFD) Liver
Muscle
↓ACC, FAS and SREBP-1⇒
Downregulated lipogenic genes. No
effect on C/EBPα, PPAR-γand LPL.
↑CD36, ACOX1, ATGL, HSL⇒↑ Lipid
uptake, oxidation and lipolysis. No effect
on CPT1, PPARα.↑pAMPK/AMPK.
↓BIP and PDI (muscle) ⇒ ↓ endoplasmic
reticulum stress
[54]

Nutrients 2020,12, 2050 15 of 28
Table 4. Cont.
MO Material Animal Model
Organ or
Biological
Sample
Evidences
MO Treated vs. Untreated Ref.
Aq MO Diabetic Liver Normalized gene expression: ↑GS, ↓PC
and caspase 3 [46]
Niazirin (seed) Diabetic
(db/db) Liver
↑HK and PK enzymes (glycolytic) and ↓
G6Pase and PEPCK (gluconeogenic).
↑
PPAR-
α
,
↓
SREBP-1 and FAS expression.
↑ratio P-ACC/ACC.
[67]
Extracts are from leaves unless specified in brackets; MO: M. oleifera; ME MO: methanolic extract of MO; FM:
Fermented MO leaves; NFM: Non-fermented MO leaves; Et MO: Ethanolic extract of MO; Aq MO: Aqueous extract
of MO; MOP: M. oleifera powder; HFD: High-fat diet; VHFD: Very-high-fat diet; H&E: Hematoxylin and eosin; TC:
Total cholesterol; LDL-C: Low-density lipoprotein cholesterol; HDL-C: High-density lipoprotein cholesterol; TG:
Triglyceride; NEFA: Non-esterified fatty acids; STZ: Streptozotocin; GOT: Aspartate aminotransferase; GPT: Alanine
aminotransferase; NAFLD: Non-alcoholic fatty liver disease; FAS: Fatty acid synthase; SREBP-1: Sterol regulatory
element-binding protein; FSP27: Fat-specific protein 27; ATGL: Adipose triglyceride lipase; ACC: Acetyl-CoA
carboxylase; EBP
α
: Enhancer-binding protein alpha; PPAR
α
: Peroxisome proliferator-activated receptor alpha; LPL:
Lipoprotein lipase; CD36: Cluster of differentiation molecule 36; ACOX1: Peroxisomal acyl-CoA oxidase 1; ATGL:
Adipose triglyceride lipase; HSL: Hormone-sensitive lipase; AMPK: 5
´
-AMP activated protein kinase; pAMPK:
phosphorylated AMPK; BIP: Binding immunoglobulin protein; PDI: Protein disulfide isomerase; GS: Glycogen
synthase; PC: Pyruvate carboxylase; HK: Hexokinase; PK: Pyruvate kinase; G6Pase: Glucose-6-phosphatase; PEPCK:
Phosphoenolpyruvate carboxykinase. ↑: Higher; ↓: Lower.
3. Scientific Evidences of M. oleifera’s Effect on Glucose Control in Animal Models
Many studies have investigated the hypoglycemic effect of different parts of MO in animal models,
mainly the aqueous or methanolic extracts of leaves and, secondly, seeds. Dry leaves have also been
employed as powder. These studies are usually performed in streptozotocin (STZ) or alloxan-induced
diabetic rats and a fewer other studies have been performed in obese animals fed high-fat diets (HFD).
The three of them are used as models of DM. The first two models rely on chemical destruction of
pancreatic
β
-cells. Both chemicals are employed as cytotoxic glucose analogues that tend to accumulate
in pancreatic
β
cells through glucose transporter 2 (GLUT2). However, depending on the dose of
chemical employed, usually through intraperitoneal injection, more resemblance to Type-1 or Type-2
diabetes is observed [
77
,
78
]. Moreover, there is some controversy as to whether alloxan and STZ
can be used to create type-2 diabetes models since both exert toxicity on
β
-cells (alloxan through
reactive oxygen species [ROS] formation and STZ through DNA methylation that causes a chain of
damages leading to DNA fragmentation) instead of inducing insulin resistance, which is the main
characteristic in type-2 diabetes. Only at low dose and in combination with HFD [
79
] or when used
with neonatal rats that will develop hyperglycemia in the adult age [
36
], can STZ be considered to
induce type-2 diabetes [
77
,
78
]. This is an important point that has not been consistently approached in
the experiments performed and the corresponding published works.
In animal models of diabetes, different approaches have been used to administer MO to the
animals or incorporate it to the diet. Mainly two ways have been used, either delivery of any type of
previously prepared extract in aqueous solution through oral gavage or incorporation of dry material
(either lyophilized extracts or blended dry leaves) to the normal diet. As an example, Khan et al. [
43
]
prepared an aqueous extract, obtained through maceration of leaf powder during approximately 24
h, with continuous stirring and then filtration and lyophilization to get a solid residue that is later
incorporated to the standard diet.
Another type of procedure is the extraction with organic solvents (n-hexane plus methanol,
ethanol, etc.) and evaporation to obtain a solid residue, preserved by freeze drying and storage,
which is then reconstituted in distilled water previously to the oral administration, commonly through
gavage [
36
,
58
]. Regarding extract composition, some protocols have been published to standardize
extract production in order to control dose and activity of the ingested active components of those
extracts [
10
,
56
]. These can be helpful techniques to characterize products in nutraceutical development.

Nutrients 2020,12, 2050 16 of 28
However, the most frequent form of MO consumption so far, is as a food, mainly fresh or dry leaves,
both for animal feeding and human consumption.
3.1. Experiments with Raw M. oleifera Leaves or Seeds
Three animal studies have been published investigating the hypoglycemic effect of MO using
dry leaf powder. In Villarruel-L
ó
pez et al. study [
40
], the administration of 50 mg/day MO dry leaf
powder during 8 weeks to alloxan-induced diabetic rats led to a decrease in blood glucose, measured
by an Accu-Check Active
®
device (Roche Diagnostics
®
, Indianapolis, IN, USA) at week 2, which
tended to remain on later weeks. In this study it is not clear whether glucose was measured in the
fasted state since fasting is only explicitly mentioned prior to sacrifice. Similar results were reported
by Oboh et al., [
41
] in STZ-induced diabetic rats who received 2% or 4% MO leaf in their diet for
14 days, with or without acarbose, an
α
-glucosidase and
α
-amylase inhibitor. Both doses (2% and 4%)
progressively and significantly reduced fasting blood glucose during treatment. In the third study,
an acute glucose intolerance ameliorating effect of the MO dry leaf powder was found. This study
consisted of an oral glucose tolerance test (OGTT) performed in spontaneously diabetic Goto-Kakizaki
rats receiving a single oral dose of glucose (2 g/kg b.w.) plus MO (200 mg/kg b.w.) compared to diabetic
rats that received only glucose [
39
]. This effect was also shown to some extent in normal Wistar rats
not suffering from diabetes [39].
In Oboh et al. study [
41
], when seeds were used instead of leaves, the reduction in fasting blood
glucose level was also significant. However, 4% MO leaves in the diet (with or without acarbose)
showed the most reducing effect compared to the other leaf (2%) and seed (2% and 4%) supplements.
In a different study that also used MO seeds powder, at doses of 50 and 100 mg/kg b.w. mixed with the
diet, reductions of 35% and 45% in fasting blood glucose and 13% and 22% in glycated hemoglobin
(HbA1C) were observed after a 4-wk treatment compared to the positive control group [42].
3.2. Experiments with M. oleifera Aqueous Extract
3.2.1. Acute Effects
Regarding acute hypoglycemic effects, evidence was obtained using leaf aqueous extracts, instead
of the powdered leaves, both, with or without oral glucose challenge [
43
,
44
]. In the first case,
STZ-induced diabetic rats pretreated with 200 mg/kg leaf extract 90 min before an oral glucose
challenge showed significantly lower glucose levels at 1, 2 and 3 h post-challenge [
44
]. In this study, the
100 mg/kg dose had a smaller effect and the 300 mg/kg dose showed a similar effect than the 200 mg/kg
dose, which decreased around 25% the glucose values two hours post-glucose. In this respect, other
authors using the same diabetes induction model and a dose of 100 mg/kg of the extract observed
approximately 50% reduction in 2-h post-challenge blood glucose compared to the control group [
43
].
Finally, in normal Wistar rats, the hypoglycemic effect during an OGTT performed after 30 min of
being gavaged with 20 mL/kg MO leaf tea was studied [
45
]. An overall decrease of 18% in postprandial
glucose was observed in the 150 min following glucose challenge.
The acute anti-hyperglycemic effect was also shown with MO aqueous leaf extract in STZ-diabetic
rats which had blood glucose measured (without oral glucose challenge) at 2-hourly intervals after
100 mg/kg extract administration in 2 consecutive days [
43
] and in normal Wistar rats 6 h after
200 mg/kg extract administration on a single occasion [
44
]. In Khan et al. study [
43
], diabetic treated
rats showed a maximum fall of 53.2% in fasting glucose after 4 h of oral administration. Similar effects
were evidenced in mice whose diabetes was induced through a HFD, showing a 34% decrease in blood
glucose on day 2 that normalized compared to control (non-diabetic) mice and more than 50% decrease
on day 3 [43].

Nutrients 2020,12, 2050 17 of 28
3.2.2. Long-Term Effects
The MO aqueous leaf extract has also shown a chronic hypoglycemic effect in experiments with
long-term treatments. In example, this effect was proven in STZ-diabetic rats along three weeks of
intervention with 100 mg/kg [
43
] or 200 mg/kg [
44
] aqueous extract compared to STZ-diabetic rats
untreated with the extract as well as in HFD-induced diabetic mice, the last ones showing complete
normalization in 7 days [
43
]. The chronic lowering effect of hyperglycemia was also observed in
alloxan-induced diabetic rats orally administered MO aqueous extract (250 mg/kg or 300 mg/kg) for
18 days [
46
,
47
] or 24 days [
48
] compared to the blood glucose levels in the control group at the end of
the intervention, and in a similar mice model administered with a dose of 100 mg/kg aqueous extract
for 14 days [
49
]. In the last one, both, fasting blood glucose and HOMA-IR were significantly improved
compared to diabetic untreated mice. Following similar methods but administering the aqueous MO
extract (200 mg/kg) for a longer period that extended for 8 weeks, a 62% reduction in fasting glucose
was found in STZ-induced diabetic rats [
50
], while a lower dose (100 mg/kg) of a similar extract led to
a 33% reduction of blood glucose levels over a period of 24 weeks [
51
]. Further, in a 12-week study,
with several OGTT performed over the course of the intervention, the control of blood glucose levels
after the oral challenge was confirmed. This C57BL/6J mice model used very high-fat diet with 5% MO
leaf concentrate of an aqueous extract providing similar doses of 200 mg MO/kg/day. The area under
the curve (AUC) was significantly lower than in the control diabetic animals for the OGTT performed
at weeks 8 and 12 (but not at 4th week of treatment) [
28
]. Mixed findings have been reported with
a supplementation (300 mg/kg) lasting 4 weeks in high fructose diet-induced diabetic animals, with
a non-significant decrease in glucose (from 133 to 129 mg/dL, mean values) but normalization of
hyperinsulinemia (from 5.05 to 2.64 µIU/mL, mean values) [52].
Moreover, experiments have been performed with a leaf protein isolate obtained by aqueous
extraction, precipitation and dialysis [
53
]. When used at concentrations of 300 mg/kg and 500 mg/kg
(but not 100 mg/kg), intraperitoneal administration in alloxan-induced diabetic mice significantly
decreased blood glucose after a single-dose, as well as after a daily dose for 7 days. However, no
effect was shown when administered orally, which the authors suggest might be explained by the
gastrointestinal digestion of ingested proteins. However, this would require that other bioactive
components of the leaf also exert hypoglycemic activity since oral administration is the preferred route
and one that has shown significant effect on glycemia in many studies. The hypoglycemic effect of
the intraperitoneally administered protein isolate was not accompanied by insulin increase, which
suggests that the effect is not caused by stimulation of insulin secretion [53].
3.3. Experiments with M. oleifera Methanolic or Ethanolic Extracts
In the case of the experiments performed in animal models with organic solvent extracts, it is
necessary to point out that leaves, seeds, pods and bark have been employed as raw material, which
may greatly influence the amount of bioactive substances in those extracts. However, due to similarity
in experimental model designs they are reported together.
3.3.1. Acute Effects
Oral administration of MO ethanolic extract at 500 mg/kg altered the hyperglycaemic condition of
fasted, STZ-induced Type-2 diabetic rats after 90 min compared to the control group [
36
]. This extract
also showed a significant effect on glucose tolerance one and two hours post oral glucose challenge.
At these time points lower blood glucose levels were found in treated animals. However, there was no
significant increase in plasma insulin levels at different times during the 2 h following a single dose
of an ethanolic extract from plants cultivated in Bangladesh [
36
]. These authors assayed potential
methods implicated in
in vivo
and
in vitro
assays and concluded that glucose absorption was probably
reduced as a consequence of reduced
α
-amylase activity which led to lower carbohydrate digestion,
while insulin secretion was not involved.

Nutrients 2020,12, 2050 18 of 28
3.3.2. Long-Term Effects Measured through Glucose Oral Challenge
A hypoglycemic effect was observed in HFD-fed mice when comparing those treated with
fermented MO leaves methanolic extract (250 mg/kg) for 8 weeks and untreated mice [
54
]. On the
contrary, no differences were found when mice were treated with non-fermented MO extract.
The fermentation was made with three Lactobacillus strains from cabbage kimchi and the treatment
with the methanolic extract resulted in lower blood glucose levels at 60, 90 and 120 min during an
OGTT performed at the end of the study. Another experiment in response to glucose challenge,
intraperitoneal this time, resulted in lower glucose levels during the 2 h following glucose injection in
alloxan-induced diabetic rats that had received a MO leaves methanolic extract (300 or 600 mg/kg)
daily during 6 weeks by oral gavage (Nigeria cultivation) [
55
]. The results were similar to the effect
of metformin. AUC for glucose was more than 50% reduced with the two doses of the methanolic
extract and with metformin compared to the untreated diabetic rats. Alongside, the methanolic extract
improved insulin release [
55
]. A different study, performed with MO seeds ethanolic extract (161 mg
MO isothiocyanate [MIC-1]/kg) showed significant blood glucose reducing effects in OGTT performed
at weeks 2nd, 4th, 6th, 9th and 12th of treatment in mice with very-high fat diet-induced diabetes
compared with untreated diabetic animals [56].
Regarding postprandial insulin levels in plasma, the different results observed with the organic
solvent extracts of MO leaves can be explained by the different experimental designs. Firstly, different
chemicals were used to induce diabetes and at different rodent’s age, i.e., adult rats [
55
] compared
to neonatal rats [
36
], respectively, and secondly, the MO extracts were given during very different
periods of 6 weeks [
55
] and single dose [
36
] in each of the experiments. It cannot be ruled out that the
ethanolic extract could induce insulin release with longer periods of treatment in STZ-induced diabetic
rats [
36
]; however, and thirdly, the different origin of the MO plants, i.e., Nigeria vs. Bangladesh, and
the extraction methods employed can also influence the underlying hypoglycemic mechanisms due to
the different composition of the leaves and extracts.
3.3.3. Long-Term Effects Measured through Fasting Blood Glucose
There is a substantial number of animal studies assessing the effect of MO extracts obtained
with organic solvent extractions on blood glucose levels after days or weeks of treatment. The fasted
conditions of the animals at blood withdrawal is the general rule in these studies, although in certain
works this fact is not clearly stated. In this sense, lower fasting blood glucose levels were found in
both STZ-induced diabetic rats and normal rats after administration of 250 mg/kg MO methanolic
extract (Nigeria cultivar) daily for 6 weeks compared to their respective control rats [
57
]. Coincident
results were found in a similar model in two different works with Indian MO; the first one an 8-wk
intervention with the methanolic extract (300 mg/kg) [
59
] and the second one with a methanolic extract
from pods instead of leaves (150 mg/kg or 300 mg/kg), which was orally administered for 3 weeks [
60
].
An increase in insulin was also noted in both of these experiments. Lower fasting blood glucose and
increased insulin was also found in db/db mice after oral administration of an ethanolic extract of MO
leaves from Cambodia (150 mg/kg) for 5 weeks (from 483 to 312mg/dL) [
62
]. Fasting blood glucose
was also lower in alloxan-induced diabetic rats that had received an ethanolic extract (200 mg/kg;
Nigeria cultivation) twice daily for 5 days compared to those measured in the control group [
63
].
A different experiment confirmed this finding in the same animal model but treated with an ethanolic
extract of MO stem bark daily (250 mg/kg, India cultivar) for one week [
64
]. Opposite to all these
positive results, the study by Olurishe et al. [
65
] failed to find a long-term improvement in fasting
blood glucose with animals receiving an ethanolic extract (300 mg/kg) of MO leaf (Nigeria cultivar), in
this case during 6 weeks. Improvements were found until day 21st compared to the hyperglycemic
levels measured on day 1st, both in the MO treated group and in the group receiving a combined
treatment with MO extract and sitagliptin (an anti-diabetic agent); however, the beneficial effects were
not maintained at 35th and 42nd days of treatment. On the contrary, end-of-study glucose levels were
significantly lower than day 1st levels if rats were treated with sitagliptin alone, thus indicating that

Nutrients 2020,12, 2050 19 of 28
the combined treatment had a less antihyperglicemic effect, may be due to drug-herb interactions. In a
similar experiment, a 24-day long intervention with ethanolic and methanolic extracts form MO leaves
(Nigeria cultivar) performed in alloxan-induced diabetic rats, led to significant reductions of around
70–85% in fasting blood glucose compared to diabetic control rats [48].
The study by Raafat and Hdaib [
66
], in alloxan-induced diabetic mice given intraperitoneal
injections of n-hexane extract of MO seeds (40, 60 or 80 mg/kg) for 8 consecutive days reported a 55%,
62% and 70% decrease in blood glucose, respectively, on the 8th day. Blood glucose was measured
6 h post injection every other day. A similar effect was also observed when mice were injected with
a chromatographic fraction of this extract identified by GC-MS as
β
-sitosterol. Both, MO extract
and
β
-sitosterol, led to significantly higher levels of insulin 8 weeks post-administration than in the
diabetic control animals. This long-term insulin-secretagogue effect seems to be involved in glycemic
homeostasis and in the lower levels of HbA1C also found in treated mice 8 weeks post-administration.
Another study with a seed’s purified compound is that of Bao et al. [
67
]. A 95% pure Niazirin
compound from concentrated aqueous extract of MO seeds was tested for its anti-hyperglycemic
and anti-inflammatory effects in db/db mice being administered 10 or 20 mg/kg/day (oral gavage)
for 4 weeks. This long-term study showed a significant decrease in fasting blood glucose compared
to untreated animals and reduced insulin levels with improved HOMA-IR. Moreover, in an OGTT,
significantly lower blood glucose was observed after 90 min and 120 min [
67
]. The lowering effect of
Niazirin on blood glucose of diabetic rats had been previously observed by Wang et al., who have also
documented similar effects with other phenolic glycoside compounds isolated from MO seeds and
administered intravenously during 2 weeks [68].
The majority of long-term studies found positive results on fasting glucose and OGTT, however,
the effect on insulin levels did not so such coincidence among studies. While some of them report
an increase in fasting insulin levels others report a decrease [
67
] or no effect [
36
,
49
,
53
]. Although the
studies are performed using different types of material, and different lengths of treatment, there does
not seem to be a clear pattern leading to an explanation for the varying results. Lack of consistency is
also found regarding the effect of MO methanolic or aqueous extracts on fasting blood glucose when
administered to normal rats. Some studies report decreased values [
58
] while more frequently, works
report no changes [
40
,
43
,
49
,
59
], although, once again, the intervention period has different durations.
4. Scientific Evidences of M. oleifera Effect on Glucose Control in Human Studies
Contrary to the abundant number of animal studies reviewed above, there is a paucity of
published clinical trials in humans, and especially those which include an adequate number of subjects.
In addition, relevant methodological data are missing in some of them. The eight human studies with
glucose control measurements found in the literature are presented in Table 2. Two studies have been
performed with administration of capsules/tablets of ground MO leaves to non-insulin dependent type
2 diabetic patients who received only oral anti-diabetic medications and dietary recommendations
to reduce energy intake. In the first study, by Kumari et al. [
72
], 22 diabetic patients received MO
treatment and nine did not, while in the second study, by Giridhari et al. [
73
], 60 patients were divided
equally into two groups (MO treatment and control). The duration of the intervention was 40 and
90 days, respectively. In Kumari et al. study, daily intake of 8 MO g led to 26% decrease of postprandial
glucose at the end of the intervention [
72
]. Meanwhile, in Giridhari et al. study, two tablets per day
of an unknown amount of MO powder, reduced postprandial blood glucose level from 210 mg/dL
to 191, 174 and 150 mg/dL, respectively, after the first, second and third month of supplementation
(29% decrease) [
73
]. In this last study, HbA1C was also reduced after treatment (from 7.81
±
0.51%
to 7.40
±
0.63%) while this was not the case in the control group. However, basal differences were
observed between MO and control groups both in HbA1C and initial postprandial glucose, which
decreases somehow the resulting evidence grade since the subjects under MO treatment started with a
worse glucose control than the other group.

Nutrients 2020,12, 2050 20 of 28
In another study, 60 postmenopausal, but otherwise healthy women, divided into two parallel
groups, received, either, no supplement or 7 grams of MO leaves powder, respectively, during
3 months [
71
]. In this period, 13.5% decrease in fasting glucose was observed. Moreover, the number
of women normalizing glucose values (i.e., <110 mg/dL) in the MO group was higher than in the
control group. Some of these studies also report significant reductions in serum cholesterol and
tryacylglyceride levels [
72
] and improvements in the level of antioxidant vitamins (retinol and ascorbic
acid) and antioxidant exzymes (superoxide dismutase [SOD], glutathione peroxidase [GSH-Px]) and
oxidative stress biomarkers (malondialdehyde [MDA]) [71].
A randomized-placebo controlled study was performed by Taweerutchana et al. [
70
], including
32 therapy-naïve DM patients whose glucose control was evaluated while undergoing a short-term
therapy (28 days) with eight daily capsules of either MO (4 g/d) or placebo. The volunteers performed
9-point glucose measurements weekly with a glucometer, which included premeal and postmeal
measurements. The results showed no difference in fasting plasma glucose, HbA1C, mean daily
plasma glucose, mean premeal, and mean postmeal plasma glucose between MO leaf and placebo
group. A non-significant reduction of 0.2–0.3% was found in HbA1C as compared to baseline in both
treatment arms, which reflected that self-monitoring plasma glucose provides feedback that improves
glycemic control through lifestyle changes. The same dose (4 g) of a similar manufactured product of
MO leaf had previously shown in 10 healthy volunteers an increase in mean insulin secretion compared
to placebo [
74
]. Insulin was measured at fixed intervals during six hours after MO ingestion, while
otherwise fasting condition was maintained. The mean plasma insulin in MO and placebo groups
were 4.1
±
7.1 and 2.3
±
0.9
µ
U/mL, respectively and the AUC of the insulin/glucose ratio was 74%
higher in the MO group. However, fasting blood glucose was similar in both groups and always within
the normal range. Since a similarity has been reported between proteins isolated from MO leaf and
insulin [
53
], the increase in insulin secretion might be explained by cross-reactivity of the antibodies
used in the immunoassay with these MO proteins or the peptides resulting from their gastrointestinal
digestion. In addition, the lack of effect on fasting plasma glucose might be explained by the fact that the
subjects were healthy and fasted, and the homeostatic mechanisms are expected to effectively prevent
hypoglycemic effects in them. On the other hand, considering the more physiological 9-point study in
DM patients [
70
], the discrepancy with the observations in animal models showing a glucose tolerance
improving effect might be attributed to too small amounts of some of the bioactive compounds, such as
moringinine or chlorogenic acid [
70
]. Thus, among the three studies performed with diabetic patients
taking MO for 28, 40 and 90 days, respectively [70,72,73], none of them has clearly shown differences
in blood glucose between treatment and control groups. However, changes from baseline have been
reported in some of them in different parameters such as fasting blood glucose [
72
] or postprandial
glucose [
72
,
73
] or HbA1C [
73
]. These findings should be pondered by bearing in mind that both of
these studies have methodological flaws, such as a low number of subjects [
72
] or apparent differences
in basal values between treatment and control groups [73].
In this respect, a higher dose, in the form of 20 g of MO dried leaf power administered as part of a
meal, resulted in lower postprandial glucose levels compared to a control meal in a group of Saharawi
diabetic patients [
12
]. Also, the increase in glucose from baseline was lower at 90, 120 and 150 min
after the beginning of the meal. In contrast, healthy adults did not show any difference in postprandial
glucose [
12
]. These findings suggest that MO could be useful in improving the glycemic response in
populations which have no access to drug therapies. Another single dose study, performed in healthy
adults who were administered MO tea has revealed that the lower dose of 200 mL induced a higher
reduction in glucose levels 30 min after glucose overload (22.8%) compared to the higher dose (400 mL,
17.9%) [
45
]. However, the final glucose levels at 150 min were similar between treatment groups and
significantly lower compared to the control group. These findings suggest the potential anti-glycemic
effect of MO in healthy adults and provide new insights about the different impact and mechanism of
action of different doses of MO, suggesting that the lower dose had a more potent effect on intestinal
glucose absorption while the higher dose had a higher effect on circulating glucose [
45
]. In addition,

Nutrients 2020,12, 2050 21 of 28
the intake of cookies containing MO leaf powder revealed a significant blood glucose reduction 30
and 45 min after ingestion compared to control cookies, as well as a decrease in hunger ratings [
69
].
Based on the studies described here, the evidence in humans on the potential usefulness of MO as an
adjuvant therapy to control hyperglycemia is so far limited but promising, especially that coming from
postprandial studies. There is, however, partial inconsistency and methodological weaknesses in some
of the published studies.
5. Dose Comparison between Animal and Human Studies
Comparing the doses employed in animal and human studies could help devise future steps in
research. When considering the amount in the three diabetic animal experiments published using
MO dry leaf powder as test material (Section 3.1) is necessary to pay attention to the administration
procedure. The first study [
40
], via oral cannula needle, administered 50 mg/day, which, using the
guide for dose conversion between animal and human by Nair and Jacob [
80
] would equate to 4.3 g
for an 80 kg human. The second experiment in rats [
39
] administered 200 mg/kg b.w., equivalent to
2.6 g/d for human. The third experiment [
41
] using 2 or 4% MO leaf powder mixed in the diet, would
approximately amount to 2650 and 5300 mg/kg b.w., respectively, if 20 g of diet are consumed per day,
which for a human would equate to 34 g/day and 68 g/day. This amount of dry leaves could be in the
upper range of a safe intake since there is a recommendation not to exceed 70 g of fresh leaves per day
in long term consumption [
81
]. However, the actual grams eaten per day by the rats in the experiment
is not reported [
41
]. Dry leaves have a higher concentration of phytochemicals and nutrients (except
vitamin C) than fresh leaves per 100 g of matter [
4
,
82
]. Although antinutrients might be an issue
here, MO has been found to contain a relatively low amount of them (phytates, saponins, tannins
and oxalates) [
83
]. Overall, it seems that doses used in animal studies that have shown hypoglycemic
effects in diabetic rats are within the physiological range. However, most of the experiments in animals
have been performed with dried (or freeze-dried) leaves extracts. In humans, with the exception of
the published work of Fombang and Saa [
45
] in 15 healthy individuals who consumed a hot MO leaf
powder tea (200 mL and 400 mL or control; 5 subjects per group) prior to a OGTT, no clinical trials
have reported effects of aqueous or alcoholic preparations of MO leaves. A typical dose of an extract in
rodent’s experiments is around 200–300 mg/kg b.w. and no adverse effect have been observed with a
methanol extract up to a dose of 3000 mg/kg [
84
]. Thus, it seems possible to obtain positive results
for glycemic control with physiological doses of the extracts in animal experiments. Translating this
to humans would require well powered human studies to evaluate the dose-effect responses within
human adjusted ranges as well as, simultaneously, providing evidence that no safety issues arise with
these preparations. According to Fahey et al. [10]; a cold tea preparation (8 ounces or 227 mL) of MO
chopped or powdered leaves contains little protein but enough glucomoringin (200
µ
mol or more;
at least 25% of the hot water molar yield of glucomoringin) and also myrosinase activity to provide
significant biological activity through conversion of the former to the isothiocyanate moringin. On the
contrary, hot teas prevent this conversion because of the inactivation of the heat-sensitive enzyme.
In this case, the bioavailability of isothiocyanates from glucosinolates would rely on the unique gut
microbiome activity of each individual. Thus, performing clinical trials with cold water extracts of MO
leaves has been encouraged [10] and should be exempt of safety issues at reasonable intake levels.
6. Scientific Evidences of M. oleifera Effect on Metabolic Alterations Related to Diabetes
Induction of diabetes is a convenient model to study not only glucose control but also other
parameters that are altered as a result of diabetic complications, such as those resulting from
increased hyperglycemia-induced oxidative stress. Thus, serum lipid profile, antioxidant enzymes and
lipid peroxidation, inflammatory cytokines and microscopic lesion observations of different tissues
and organs have been measured in many diabetes-induced animal studies. These parameters are
associated with the neuropathy, nephropathy, retinopathy, cardiovascular and other pathophysiological
manifestations in diabetic disease [42,51,57].

Nutrients 2020,12, 2050 22 of 28
As shown in Table 1, and in more detail in Tables 3and 4, scientific works are accumulating
in which an extended number of pathophysiological manifestations that frequently coexist with
glucose intolerance or diabetes are studied. For the sake of systematization these are divided in
two wide areas in this review, namely, (1) antioxidant capacity and inflammation (Table 3), and (2)
lipid accumulation, histopathology of damaged organs and gene expression of proteins involved in
metabolic pathways (Table 4). However, diabetes complications originate from a complex interrelated
system of cellular and metabolic mechanisms which are not independent from one another. One of
them is the increased production or ineffective scavenging of ROS [
85
]. One outcome of excessive levels
of ROS is the modification of the structure and function of cellular proteins and lipids, leading to overall
dysfunctional biological activity, immune activation and inflammation [
86
]. In this sense, numerous
studies have consistently shown that different parts of MO, mainly leaves [
41
] and seeds [
41
,
42
],
but also pods [
60
] have potent antioxidant capacity in animal models of diabetes. Indeed, these parts
of MO are able to repair the high levels of oxidative stress that are intrinsic to the chemical induction
of diabetes in the animal models and in animals fed a very high-fat diet. This effect has also been
observed with plant extracts, both aqueous and obtained with organic solvents, and documented in
many different organs, such as the liver [
46
,
52
,
61
], kidney [
57
], pancreas [
50
], brain [
76
] and heart [
59
].
The election method employed to test the antioxidant capacity in these organs is the measurement
of lipid peroxidation through the formation of MDA, which is consistently diminished in diabetic
animals treated with MO compared to untreated animals. This might be explained by the effect of
MO on the activity of antioxidant enzymes, since many study results confirm that MO and its extracts
can reverse the decrease in SOD, catalase (CAT) [
42
,
46
,
52
,
59
,
60
,
76
] and GSH-Px [
41
,
59
] observed in
diabetic animals compared to the negative control, as well as increase the non-enzymatic antioxidant
system GSH (reduced glutathion) [
41
,
42
,
46
,
50
,
59
–
61
], which is also decreased in the diabetic condition.
The results, as shown in Table 3, are very consistent among studies and in all type of organs; only
Paula et al. study [
53
], performed with a protein isolate of MO leaves, showed increase in CAT but no
effect on SOD activity, perhaps due to a short treatment duration of only 7 days. On the other hand,
controversy also seems to arise when normal control rats instead of diabetic rats are used to test the
methanolic extract of MO. In this case, no significant differences were observed in the levels of SOD,
CAT, GSH-Px and GSH although consistent trends pointed towards higher levels of these antioxidant
molecules [59].
Regarding inflammation, positive effects of the oral administration of MO and its extracts have
been observed when measuring the expression of inflammatory cytokines in the liver and muscle [
54
],
kidney [
62
] and wound tissue [
75
] of diabetic animals. Similar results were found when measuring the
concentration of inflammatory cytokines in liver [
58
], kidney [
42
,
57
], and plasma or serum [
28
,
42
,
67
].
Specifically, this reduction of inflammation has been observed on TNF-
α
, IL-6 [
28
,
54
,
58
,
62
,
75
] and
iNOS [
56
,
62
,
75
] levels. In addition, lower concentrations of other inflammation markers such as
IL-1
β
[
28
,
62
,
75
], MCP-1 [
58
] and COX-2 [
62
,
75
] were also found. On the opposite, an increase in the
anti-inflammatory cytokine IL-10 [
67
] and also in IFN-
γ
production by peripheral blood mononuclear
cells [49] have been described in MO supplemented diabetic mice.
Other extensive information derived from the same experimental models and interventions
with fresh, dried or variably processed parts of the plant MO, evidences that it has an effect in
the structural and functional protection of different organs that can be partially evaluated through
histological analysis of tissue sections (Table 4). In this sense, Omodanisi et al. [
58
] observed that
oral administration of a methanolic extract of MO (250 mg/kg) to STZ-induced diabetic animals for
six weeks induced a significant reduction in altered hepatic enzyme markers compared to untreated
diabetic rats and the hepatoprotective effect was also proven with the histopathology analysis of
liver sections. Reduction of hepatic enzymes was also observed by Khan et al. [
43
], as well as the
prevention of liver histopathological changes in a number of different studies [
46
,
67
]. Improvement
in histopathological findings associated to diabetes induction have also been documented in renal
tissue [
62
], pancreas [
42
,
46
] and heart [
59
]. Lower hepatic lipid accumulation has also been evaluated

Nutrients 2020,12, 2050 23 of 28
through microscope observation in response to fermented MO [
54
], and in homogenate quantification
after administration of extracts from seeds [56], leaves [28] and niazirin [67], respectively.
Regarding the analysis of the serum lipid profile, the results seem to be consistent about the
hypolipidemic effect of MO. A significant decrease of LDL-cholesterol and triacylglycerides are
reported in different works comparing treated and untreated diabetic animals [
43
,
55
,
62
,
67
]. Some
of them also find decreased total cholesterol levels [
43
,
55
,
58
] and increased HDL-cholesterol with
MO extracts [
43
,
55
] and MO seeds-purified niazirin [
67
]. Khan et al. [
43
] observed a smaller level of
restoration of lipid profile in STZ-induced diabetic mice than in HFD-induced diabetic mice.
Finally, efforts have been also made to shed light into the molecular mechanisms that lead to lower
lipotoxicity and lipid uptake during treatment with MO in diabetic models. In this line, Joung et al. [
54
],
measured the expression of genes involved in liver lipid metabolism and found a decreased expression
of the lipogenic genes ACC, FAS, SREBP-1 with fermented MO leaf and increased lipolytic gene
expression as indicated by CD36, ACOX-1, ATGL and HSL measurements. No effect, however, was
observed on the expression of the lipogenic or lipogenesis regulatory genes C/EBP
α
, PPAR-
γ
, LPL and
pAMPK and the lipolytic CPT1 and PPAR-
α
. They also found decreased endoplasmic reticulum stress
in skeletal muscle as shown by lower expression levels of the protein chaperon BiP and the disulfide
bond regulator PDI under MO treatment of the diabetic animals. Coincident findings regarding reduced
SREBP-1 and FAS expression were found by other authors [
67
] who also reported an increase in fatty
acid oxidation as documented by increased ratio p-ACC/ACC in the liver of db/db mice treated with
niazirin from MO. Since lipotoxicity and insulin resistance are closely related as metabolic abnormalities
present in diabetes development, the expression of genes involved in glucose homeostasis has also been
studied. Abd Eldaim et al. [
46
] found that a MO aqueous extract increased the expression of glycogen
synthase gene (glycogenic), decreased that of pyruvate carboxylase (gluconeogenic) and the apoptotic
caspase 3 gene in liver, which prove the normalization exerted by MO treatment on the dysregulated
metabolism associated to diabetes induction. This was further supported by findings showing that the
treatment with niazirin inhibited the abnormally high activity of the gluconeogenic enzymes G6Pase
and PEPCK in the liver of untreated db/db mice [
67
]. It also repaired glycolytic activity in the liver by
increasing the hexokinase and pyruvate kinase enzymes which were decreased in db/db mice [67].
7. Final Remarks
The extent of investigation on the potential use of MO plant in the control of glycaemia is still
scarce. The number of animal studies is reasonably high and covers an ample range of different
study designs trying to shed light on the observed pathophysiological changes in models of induced
hyperglycemia. A majority of the results presented show significant improvements in blood glucose,
both in fasted state and in response to a glucose tolerance test. The mechanisms of action revealed
from the animal model experiments presented here include the normalization of the gene expressions
of enzymes involved in glucose metabolism resulting in the restoration of liver glycolytic activity
and glycogen storage [
46
,
55
,
67
] as well as reducing gluconeogenesis [
28
,
46
,
67
] and improving insulin
signaling [
28
]. Delayed gastric emptying can also improve glycemic control in the postprandial
state [
39
], which could be related to the high fiber content in the MO leaves. In addition, inhibition of
intestinal glucose uptake has been shown in several animal models [
36
,
39
,
43
] and has been related to
the inhibition of glucose transporter proteins in cell membranes by flavonoid glycosides [
39
]. On the
contrary, improved glucose uptake was found in muscle and liver [
43
,
55
], favoring reduction of blood
glucose and insulin-like proteins isolated from the MO plant could also contribute to the hypoglycemic
effect [
53
]. Regarding clinical trials in humans, there are only a few published studies and with very
variable designs. Thus, it is difficult to reach consensus about the indication of MO as an adjuvant
therapy in the prevention and treatment of DM. Thus far, postprandial studies seem to offer more
evidences of the hypoglycemic effect of MO leaves than the daily dose interventions lasting 28 to
90 days. More intervention studies in diabetic or prediabetic patients are certainly needed with more
stringent inclusion criteria and a sufficient number of patients. It is also highly recommended to try

Nutrients 2020,12, 2050 24 of 28
to quantify the bioactive substances administered with the experimental material tested to facilitate
comparison between studies. In this line, efforts made to characterize the composition of different
MO tree varieties and other crop influential factors will add positive knowledge to a more rational
design and use of MO food supplements. On the other hand, the use of extraction methods to obtain
preparations enriched in specific bioactive compounds requires further research before they can be
implemented for the therapy of human disease, since safety during prolonged use should be warranted
prior to this step. Nonetheless, tea preparations, especially with cold water, could provide enough
glucosinolates and its isothiocyanate metabolites for a potential biological effect, provided moringin is,
at least to some extent, responsible for the hypoglycemic effect of the plant. Finally, the composition of
the extracts and how the different ingredients interact through additive, synergistic or inhibitory effects
should be investigated because this will impact the therapeutic use of the extracted preparations or
even the isolated compounds as compared to the raw materials.
Author Contributions:
E.N.: Conception and design, acquisition of data, drafting and revising. N.R.-U.:
acquisition and interpretation of data and revising. R.M.M.-G.: acquisition of data and drafting; S.G.-M.:
acquisition, interpretation of data and revising; L.E.D.-P.: acquisition of data and revising; A.M.: conception and
revision. All authors have read and agreed to the published version of the manuscript.
Funding:
Funding: This work was performed under the frame of the NUTRIMOL-DB Project that received funding
from the Ministerio de Econom
í
a, Industria y Competitividad (MINECO), Agencia Estatal de Investigaci
ó
n (AEI)
and Fondo Europeo de Desarrollo Regional (FEDER, UE) (ref. AGL2017-86044-C2-1-R.).
Conflicts of Interest: The authors declare no conflict of interest.
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