Life 2273833

Article 1
Phytochemical profile, antioxidant, antimicrobial and antidia- 2
betic activities of Ajuga iva (L.) 3
Soukaina Saidi* 1, 2, Firdaous Remok 1, Nadia Handaq1, 3, Aziz Drioiche 1, Aman Allah Gourich1, Nawal Elmenyiy4, 4
Smail Amalich3, Mohamed Elouardi 1, Hanane Touijer 1, Mohamed Bouhrim8, Latifa Bouissane*2, Hiba-Allah 5
Nafidi5, Yousef A. Bin Jardan6, Mohammed Bourhia 7 and Touriya Zair* 1 6
1 Research team of bioactive molecules and environment chemistry, Laboratory of Innovative Materials and 7
Biotechnology of Natural Resources, Moulay Ismail University, Faculty of Sciences, Meknes, Morocco 8
2 Laboratory of Molecular Chemistry, Materials and Catalysis, Sultan Moulay Slimane University, Faculty of 9
Science and Technology, Beni Mellal, Morocco 10
3 Plant Valorization and Protection Research Team, Laboratory of Environmental Biology and Sustainable De- 11
velopment, Ecole Normale Supérieure, Abdelmalek Essaadi University, 93000 Tetouan, Morocco 12
4Laboratory of Pharmacology, National Agency of Medicinal and Aromatic Plants, Taounate 34025, Morocco 13
5Department of Food Science, Faculty of Agricultural and Food Sciences, Laval University, 2325 Quebec City, 14
QC G1V 0A6, Canada 15
6Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia 16
7Department of Chemistry and Biochemistry, Faculty of Medicine and Pharmacy, Laayoune 70000, Morocco 17
8 Laboratory of Biological Engineering, Team of Functional and pathological Biology, Faculty of Sciences and 18
Technology Beni Mellal, University Sultan Moulay Slimane, Beni Mellal 23000 Morocco. 19
* Correspondence: soukainasaidi.ss@gmail.com (SS); t.zair@umi.ac.ma (TZ); bourhamohammed@gmail.com 20
(MB); 21
Abstract: In Morocco, many applications in ethnomedicine on Ajuga iva (L.) have been recognized 22
Citation: Saidi, S.; Remok, F.;
to treat various pathologies such as diabetes, stress and microbial infections. The objective of this 23
Handaq, N.; Drioiche, A.; Gourich,
work is to carry out phytochemical, biological and pharmacological investigations on the extracts 24
A.A.; Elmenyiy, N.; Amalich, S.;
Elouardi, M.; Touijer, H.; Bouhrim, of Ajuga iva leaves in order to confirm its therapeutic effects. The phytochemical screening carried 25
M.; et al. Phytochemical profile, an- out on the different extracts of Ajuga iva showed its richness in primary (lipids and proteins) and 26
tioxidant, antimicrobial and antidi- secondary metabolites (flavonoids, tannins, reducing compounds, oses and holoside. The best con- 27
abetic activities of Ajuga Iva (L.). tents of polyphenols, flavonoids and tannins evaluated by spectrophotometric methods were found 28
in the hydroethanolic extract (69.850 ± 2.783 mg EAG/g DE, 17.127 ± 0.474 mg EQ/g DE, 5.566 ± 0.000 29
Academic Editor: Firstname Last- mg EQC/g DE) respectively. Analysis of the chemical composition of the aqueous extract by 30
name. LC/UV/MS revealed 32 polyphenolic compounds including ferulic acid (19.06%), quercetin 31
(10.19%), coumaric acid (9.63%) and apigenin-7-(2-O-apiosylglucoside) (6.8%). The antioxidant ac- 32
Received: date
tivity of Ajuga iva extracts was evaluated by three methods (DPPH*, FRAP, CAT). The hydroeth- 33
Revised: date anolic extract recorded the strongest reducing power: DPPH* (IC 50 = 59.92 ± 0.7 µg/mL), FRAP (EC50 34
Accepted: date = 196.85 ± 1.54 (µg/mL) and CAT (199.21 ± 0.37 mg EAG/gE). A strong correlation between phenolic 35
Published: date compounds and antioxidant activities was confirmed by the determination of Pearson's coefficient. 36
The antimicrobial activity of Ajuga iva studied by the microtiter method revealed potent antifungal 37
and antibacterial potency against Candida parapsilosis and Staphylococcus aureus BLACT. In vivo oral 38
Copyright: © 2023 by the authors. glucose tolerance test (OGTT) using normal rats revealed that the antihyperglycemic action of the 39
Submitted for possible open access
aqueous extract significantly reduced postprandial hyperglycaemia at (30 min, p< 0.01) and area 40
publication under the terms and con-
under the curve (AUC glucose), p<0.01. Similarly, the aqueous extract, tested on pancreatic α-am- 41
ditions of the Creative Commons At-
ylase enzyme activity in vitro and in vivo, significantly inhibited pancreatic α-amylase activity with 42
tribution (CC BY) license (https://cre-
IC50 =1.52 ± 0.03 mg/mL. In conclusion, the extract from Ajuga iva could be a good source of bioactive 43
ativecommons.org/licenses/by/4.0/).
molecules, which exhibit potent antioxidant, antimicrobial and strong antidiabetic activity, for ap- 44
plications in the pharmaceutical industry. 45
Keywords: Ajuga iva; quality control; DPPH; Frap; antimicrobial activity; OGTT toxicity; α-amylase, 46
47
Life 2023, 13, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/life

Life 2023, 13, x FOR PEER REVIEW 2 of 30
1. Introduction 48
49
Medicinal plants contain a variety of bioactive compounds that have been used for 50
thousands of years as traditional therapies for a wide range of human ailments. Nearly 51
80% of people in underdeveloped nations use medicinal plants as their primary source of 52
healthcare, according to the World Health Organization [1]. In fact, oxidative stress has 53
been associated to a number of human diseases, including diabetes, Alzheimer's, Parkin- 54
son's, cancer, and cardiovascular diseases [2–4]. Reactive oxygen species (ROS) generation 55
and the body's antioxidant defense capabilities are out of equilibrium, which leads to ox- 56
idative stress[5]. Therefore, the cytotoxic control of ROS is generally attributed to antioxi- 57
dant systems [2]. Natural antioxidants such as flavonoids, phenolics and tannins protect 58
against diseases associated with oxidative stress [6]. A decrease in antioxidants may also 59
cause the emergence of diabetic problems [7]. Persistent hyperglycemia is a hallmark of 60
the metabolic syndrome known as diabetes, which develops when the pancreas either 61
produces insufficient insulin (insulin secretion insufficiency) or when the body no longer 62
effectively uses insulin (insulin resistance) [8,9]. Additionally, more than 90% of diabetic 63
people have type 2 diabetes mellitus, a complex illness [10]. This type of diabetes stems 64
from insulin resistance, which is strongly associated with several triggering factors, in- 65
cluding poor diet, genetic predisposition and lack of physical activity [10,11]. Between 5% 66
and 10% of all diabetics have type 1 diabetes, often known as "juvenile" diabetes, which is 67
caused by the pancreas' failure to release insulin as a result of certain genetic and environ- 68
mental variables [8–10]. Modern medicine has played a crucial role in the development of 69
anti-diabetic drugs, which have significantly improved the management of diabetes. 70
These drugs significantly reduce the postprandial rise in plasma glucose and insulin levels 71
in diabetic patients by inhibiting the activity of α-glucosidases. Inhibitors of α-glucosidase 72
are often based on mimetic conformation of the substrate and/or the positive charge of 73
transition state. The inhibitors of α-glucosidase that are most involved in the digestion of 74
carbohydrate are the iminosugars like: 1-deoxynojirimycin (DNJ), castanospermine, 75
swainsonine, fagomine etc.[12,13] . But recently, it's been shown that these iminosugars, 76
along with medications like metformin and acarbose, have a lot of drawbacks like nausea, 77
bloating, weakness, etc.[11,14,15]. 78
Therefore, attention has been directed towards the use of medicinal plants to identify 79
and isolate natural bioactive molecules endowed with substantial antidiabetic effects with 80
less harmful impact on human health. One of those potent prospective medicinal plants 81
is Ajuga iva (L.), which has a long history of usage in the conventional medical system as 82
an anti-diabetic, an antioxidant, and an antibacterial. The Lamiaceae family includes the 83
herbaceous plant species A. iva (L.), which is extensively distributed in southern Europe, 84
Egypt, Tunisia, and Morocco [16]. This plant species has a number of slang names, includ- 85
ing Chendghoura and Toutoulba [17]. Some Ajuga species are regarded as weeds, yet 86
many are employed in horticulture as ground or border cover and in rock gardens [18]. 87
In Morocco, many ethno-medical uses have been reported for this plant. The aerial por- 88
tion (leaves and flowers) of A. iva has been used as a decoction to treat a variety of condi- 89
tions, including kidney and digestive disorders, diabetes, hypertension, painful menstru- 90
ation, rheumatic pain, eye infections, cardiovascular disorders, allergies, cancer and rheu- 91
matism [19–26]. Recently, several of its ethno-medical uses have been validated by stud- 92
ies, experimental scientists in laboratories, in vivo and in vitro demonstrating that the ex- 93
tracts of A. iva possess significant antibacterial, anticancer, antioxidant, antidiabetic, in- 94
secticidal, antiviral, anticancer, analgesic and antihypercholesterolemic properties [27– 95
31]. In addition, phytochemical investigations on A .iva revealed the presence of a wide 96
spectrum of chemical constituents belonging to flavonoids, tannins, terpenoids, steroids, 97
fatty acids and phenolic acids (caffeic acid and chlorogenic acid, etc.) and other principles 98
such as ajugarine C24H34O7 (MW: 392) [22,32,33]. In addition, A. iva includes essential oils 99
as well as anthocyanins, neoclerodane diterpenoids, and other compounds [34]. There- 100
fore, the pharmacological and biological activities of A. iva are mainly explained by all the 101
primary and secondary metabolites present in the leaves and fruits [20]. The purpose of 102
the current study was to support the widespread usage of A. iva (L.) by comparative phy- 103
tochemical characterizations of phenolic compounds in its extracts and evaluation of their 104
antioxidant, antimicrobial and antidiabetic effects in vitro. In addition, the antihypergly- 105
cemic activities, the inhibition of α-amylase as well as the toxicity of the decoction of A. 106
iva were performed in vivo. 107
2. Materials and methods 108
2.1. Chemicals products 109
The majority of the chemicals and solvents were bought from Sigma Aldrich. Pancre- 110
atic α-amylase and other reagents were acquired from Sigma-Aldrich, whereas acarbose 111
was bought from Bayer Schering Pharma. 112
2.2. Plant material 113

Ajuga iva leaves were gathered in June 2021 in the Masmouda region of Morocco (X= 114
471170 W; Y= 464816 N; Z= 320 m). The leaves were then air dried at room temperature in 115
the shade before being ground into a powder to create the extracts. The Scientific Institute 116
of Rabat, Morocco, carried out the plant's botanical identification. Table 1 and Figure 1 117
contain comprehensive data on this species [35]. 118
119
Figure 1. Ajuga iva (L.) (Soukaina SAIDI and Touriya ZAIR 2021). 120
Table 1. : Ajuga's taxonomic position in the family of plants [35] . 121
Reign Plantae
Division Spermatophyta
Class Dicotyledones
Order Tubiflorae
Family Lamiaceae
genus Ajuga
Species Iva
Author (L.) Schreber
122
2.3. Microbial material 123

Nine bacterial strains and eight fungus strains were used to test the antibacterial ac- 124
tivity of A. iva L. extracts (Table 2). All strains were cultivated in subcultures after being 125
revived in Mueller Hinton and Sabouraud broths (20% glycerol stock) at 80 °C. 126
Table 2. List of bacterial and fungal strains tested. 127
Bacterial strains Reference Fungal strains Reference

Staphylococcus epidermidis 5994 Candida albicans Ca
Staphylococcus aureus BLACT 4IH2510 Candida dubliniensis Cd
Streptococcus agalactiae (B) 7DT1887 Saccharomyces cerevisiae Sacc
Escherichia coli sauvage 3DT1938 Aspergillus niger AspN
Escherichia coli BLSE 2DT2057 Candida tropicalis Ct

Enterobacter cloacae 02EV317 Candida krusei Ckr
Klebsiella pneumoniae 3DT1823 Candida parapsilosis Cpa
Proteus mirabilis 2DS5461 Candida kyfer C.ky
Pseudomonas aeruginosa 2DT2138
128
129
2.4. Animals 130

Wistar rats (200-250 g) and albino mice (25-35 g) were used in the investigation of the 131
antihyperglycemic effects of A. iva decoction. The animals were raised at the National 132
Agency for Medicinal and Aromatic Plants' Department of Pharmacology's animal facility 133
in Taounate (photoperiods of 12 h of light/ 12 h of darkness at 22±2°C). The animals were 134
housed in breeding-friendly environments with unrestricted access to food and water. 135
2.5. Preparation of extracts 136
2.5.1. Preparation of the decocted extract 137

138
In a 600 mL batch of distilled water, 30 g of A. iva (L.) leaf powder was cooked for 60 139
min at 80°C with some stirring. The solution is filtered after cooling, and then it is forced 140
to evaporate. The resulting residue is subsequently dried at 60°C. The powder obtained 141
represents the decocted extract of leaves of A. iva (L.) (Ed). 142
2.5.2. Preparation of aqueous and hydroethanolic extracts 143

30 g of plant material from the leaves of A. iva (L.) were extracted with 600 mL of 144
distilled water (aqueous extract) or 600 mL of 70/30% ethanol-water (hydroethanolic ex- 145
tract) using a Soxhlet type apparatus for 12 h. In a vented oven set at 60°C, the filtrates 146
were dried. In sterile vials, the dried crude extracts of the various aqueous (EAq) and hy- 147
dro-ethanolic (Eeth) solvents were gathered and kept at room temperature until use. 148
2.6. Parameters evaluated of plant material 149
2.6.1. Water content 150

The procedure is carried out in accordance with AFNOR standard (NF-V03-402, 151
1985) [36]. The protocol consists of putting 5 g of the sample in a ventilated oven at 103 °C 152
for 24 hours until constant weight. Three times of the test were conducted, and the average 153
was calculated. The water content is measured after drying the sample by the following 154
formula (Eq1). 155
𝑴𝟎−𝑴𝟏
𝑾𝑪% = ( ) *100 (Eq 1) 156
𝑴𝟎
WC: Water content (%) 157
M0: Mass before drying (g) 158
M1: Mass after drying (g) 159
2.6.2. Ashes 160

The ash content of A. iva (L.) leaves was determined according to the standard 161
(NFV05-113, 1972) [37]. 5 g of the vegetable powder are weighed into preheated porcelain 162
crucibles. The method's basic idea is to calcine the sample at 550°C in a muffle furnace 163
until white ashes with a consistent mass are produced. The average was calculated after 164
the test was administered three times. The following formula determines the organic mat- 165
ter content: 166
𝑾 −𝑾
𝑶𝑴% = ( 𝟏 𝟐 ) ∗ 𝟏𝟎𝟎 (Eq 2) 167
𝑻𝑺
OM%: Organic material 168
W1: Capsule and sample weight before calcination 169
W2: Capsule and sample weight after calcination 170
TS: Test sample 171
Ash content is determined using the formula below: Ash % = 100 – OM %. 172
2.6.3. pH determination 173
The principle consists in bringing the quantity of vegetable matter into contact with 174
the quantity of distilled water (ratio 1:5). After filtering, the mixture is allowed to cool. A 175
pH meter with an electrode is used to measure the pH [38]. 176
2.6.4. Titratable acidity (TA) 177

10 g of the vegetable powder is brought into contact with 100 mL of boiling distilled 178
water, the whole extract is stirred for 15 min then filtered. 10 mL of the filtrate and 20 mL 179
of water are placed in a 100 mL beaker, then a few drops of phenophthalein were added 180
and the titration was carried out with a solution of NaOH (0.01 N) until a color persistent 181
pink was obtained, the volume of NaOH used was noted. This volume is converted into 182
equivalent citric acid and the titratable acidity is determined according to the following 183
relationship [39]. 184
185
weight of acid equiv.∗ normality of NaOH ∗ vol.of titration (mL)∗Dilution factor
𝑻𝑨 = 𝑠𝑎𝑚𝑝𝑙𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑖𝑛 (𝑔)
(Eq 3) 186
2.6.5. Heavy metal assays: Atomic Emission Spectrometry Coupled with Induced Plasma 187
(ICP-AES) 188
Arsenic (As), cadmium (Cd), chromium (Cr), iron (Fe), lead (Pb), antimony (Sb), and 189
titanium (Ti) are the heavy metals accused. Each metal has a contamination limit that must 190
be satisfied. Drugs whose raw components are known to accumulate significant amounts 191
of cadmium are given an exception, nevertheless. We used aqua regia (HNO3 + 3HCl) and 192
the established mineralization technique (AFNOR, 1999) to determine the key element 193
concentrations. Large sample sizes are permitted by the latter, which reduces issues with 194
sample representativeness. Crushed plant material (0.1 g) is combined with 3 ml of aqua 195
regia, which is made from 1 ml of nitric acid (HNO3; 99%) and 2 ml of hydrochloric acid 196
(HCl; 37%). The mixture is then heated in a reflux assembly at 200°C for two hours, fol- 197
lowing which it is allowed to cool and settle. The supernatant was then ta ken out, filtered 198
over a 0.45 µm membrane, and topped off with 15 ml of distilled water. The inductively 199
coupled plasma atomic emission spectrometer, or ICP-AES, was used to measure the 200
amounts of heavy metals. (Ultima 2 Jobin Yvon). 201
2.7. Analysis and identification of the chemical composition of decoction by the LC/UV/MS 202
method 203
The LC-UV-MS studies were conducted using a PLATIN LC/HPLC system that was 204
coupled to a UV detector. The mobile phase is composed of the solvent A (acetonitrile + 205
0.1% formic acid) and solvent B (water + 0.1% formic acid) mixes. The established elution 206
gradients were 0-5% B (1 min), 5-20% B (0.5 min), 20% B (3.5 min), 20-100% B (4 min), 100% 207
B (2 min), and re-equilibration 100-0% B (0.5 min), 0% B. (2.5 min). The system needed to 208
be cleaned with 70% methanol. The decoction was diluted in a 1:1 methanol/water solu- 209
tion to reach a concentration of 1 mg/ml. After that, a 2 mm PTFE filter was used to filter 210
it. For each analysis, 0.004 ml of the extract was injected. The temperature was set to 30°C, 211
and the flow rate was set at 0.3 ml/min. Based on bibliographic data, a few standards were 212
picked for this inquiry. They were injected in the exact same circumstances as a decoction. 213
Cinnamic acid, luteolin, apigenin, myricetin, ferulic acid, quercetin, vanillic acid, chloro- 214
genic acid, ascorbic acid, caffeic acid, rosmarinic acid, protocatechuic acid, gallic acid, and 215
coumarin are some of the standards in this group. 216
217
218
219
2.8. Phytochemical study of Ajuga iva (L) leaves extracts. 220

2.8.1. Phytochemical screening 221
The main chemical groups of the extracts under investigation are sought after by 222
phytochemical screening, a qualitative examination. It is based on the visual observation 223
of the color shift and/or precipitate formation following the addition of a particular rea- 224
gent. The experiments were conducted in accordance with the widely used phytochemical 225
techniques, as reported by Dey and Harborne (1989), Gibbs Darnley (1974) [40,41]. Several 226
reagents were used for the qualitative analyses. The flavonoids were identified by the cy- 227
anidin reaction. Tannins were characterized by reaction with ferric chloride. The Stiasny 228
response emphasized the difference between the two classes of tannins. Alkaloids were 229
characterized using Dragendorff's and Mayer's reagents. The detection of the reducing 230
compounds was carried out by Fehling's solution. After shaking the solution, the presence 231
of foam indicated the presence of saponins. The Liebermann-Buchard reaction was used 232
to identify terpenes and sterols. The results were given as follows: +++: strong positive; ++: 233
Moderately positive; +: Weakly positive; - : Negative. 234
2.8.2. Dosage of phenolic compounds 235
2.8.2.1. Determination of Total Polyphenol Content (TPC) 236

With slight adjustments, Singleton and Rossi's method [42] was used to measure total 237
phenolic compounds (TPC). A 50 ml volumetric flask was filled with a volume 0 <V ≤ 100 238
µl of the extract, 1.5 ml of Folin-Ciocalteu reagent (10%), and 1.5 ml of Na 2CO3 solution 239
(7.5% - w/v). The mixture was stirred and adjusted with distilled water till the gauge line. 240
241
A spectrophotometer (V-1200) was used to measure the absorbance at 760 nm follow- 242
ing incubation for 60 min in the dark. Results were expressed as milligrams of gallic ac id 243
equivalents per gram of extract (mg GAE/gE), with gallic acid serving as the standard. 244
The test was run three times, and the total concentration of phenols was determined using 245
the formula below. The tests were carried out in triplicate, and the following formula was 246
used to determine the total polyphenol content: 247
248
C∗𝑉0
𝑇𝑃𝐶 = *D (Eq 4) 249
𝑚𝑒𝑥𝑡𝑟𝑎𝑐𝑡
250
TPC : Total phenolic compounds 251
C : Concentration assessed based on the calibration curve 252
V0 : Volume of the entire extract 253
mextract : masse of extract 254
D : Dilution factor 255

Vf : Final volume to be measured in a spectrophotometer 256
Vi : Volume taken from the extract to be tested 257
2.8.2.2. Determination of Flavonoid Content (FC) 258

With a few adjustments, the AlCl 3 colorimetric method [43] was used to estimate fla- 259
vonoid content (FC). The following ingredients were added: 10 µL of the 10% (w/v) 260
AlCl3, volume 0 <V ≤ 100 µL of the extract and 2 mL of pure water. The liquid was diluted 261
to a final amount of 5 mL using just pure methanol. The absorbance was measured at 433 262
nm following a 30 min incubation period at room temperature. The FC was calculated as 263
milligrams of quercetin equivalents per gram of extract (mg QE/gE), with quercetin serv- 264
ing as the reference standard. The tests were run three times, and the FC was determined 265
as follows: 266
267
C∗𝑉0
𝑇𝐹𝐶 = *D (Eq 5) 268
TFC: Total flavonoid compounds 269
C : Concentration assessed based on the calibration curve. 270
2.8.2.3. Determination of Condensed Tannin Content (CT) 272
The condensed tannin content (CT) of the extracts was quantified using the Vanillin- 273
HCl method [44], in which a volume of each extract (<V ≤ 100 µL) was mixed with 3 mL 274
of vanillin solution prepared in methanol (4%) and 1.5 mL of concentrated hydrochloric 275
acid (HCl) (37%), and the mixture was incubated at room temperature for 20 min. The 276
absorbance was measured at 499 nm following. The extracts' condensed tannin content 277
(CT) was calculated as milligrams of catechin equivalent (mg CE/g CE) per gram of ex- 278
tract. Condensed tannin content (CT), which was determined after the analyses were per- 279
formed in triplicate, was determined as follows: 280
281
C∗𝑉0
𝑇𝐶𝑇 = *D (Eq 6) 282
T: Total condensed tannin 283
C : Concentration assessed based on the calibration curve. 284
V0 : Volume of the overall extract. 285
2.9. Evaluation of antioxidant power 287

Three distinct techniques, DPPH, FRAP, and CAT, were used to assess the antioxi- 288
dant strength of the Ajuga iva (L.) extracts. 289
2.9.1. Radical scavenging activity (DPPH*) 290

The DPPH* (2.2-diphenyl 1-picryl hydrazyl) test were carried out using Wong et al. 291
(2006) method [45] with some modifications. Briefly, 200 µL of each extract at various con- 292
centrations was added to 2.8 mL of (0.024 mg/mL) ethanol DPPH* solution. The absorb- 293
ance of the mixture was measured at 515 nm using a spectrophotometer (V-1200) after 294
incubated at room temperature for 30 min. Ethanol was used as the negative control. Bu- 295
tylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and ascorbic acid (vita- 296
min C) were utilized as the positive control. The percentage of DPPH* inhibition was eval- 297
uated using Eq. (7). 298
299
𝐴0−𝐴
𝑃𝐼% = × 100 300
𝐴0 (Eq 7)
𝑃𝐼: Inhibition of DPPH (%). 301
𝐴0 : Absorbance of free radical solution (DPPH) in the absence of the extract (Nega- 302
tive control). 303
𝐴 : Absorbance of the free radical solution (DPPH) in the presence of the extract. 304
2.9.2. Ferric Reducing Antioxidant Power (FRAP) 305
FRAP assay was performed in accordance with Oyaizu's (1986) [46] instructions. 306
Each extract was divided into an aliquot of 0.5 mL, which was then combined with 2.5 mL 307
of phosphate buffer solution (0.2 M - pH 6.6) and 2.5 mL of potassium ferricyanide (1%) 308
at various doses (ranging from 0.5 to 5 mg/mL). For 20 min, the reactions were heated to 309
50°C in a water bath. The mixture was mixed with 2.5 ml of 10% trichloroacetic acid, and 310
the combination was then centrifuged at 3,000 rpm for 10 min. 2.5 mL of the solution's 311
supernate were combined with 0.5 mL of ferric chloride solution (0.1%) and 2.5 mL of 312
distilled water. The standard utilized was ascorbic acid. A spectrophotometer (V-1200) 313
was used to measure the absorbance at 700 nm. Effective concentration of 50% (EC 50) was 314
used to express the results. 315
316
317
2.9.3. Total antioxidant activity (CAT) 318
The extracts were subjected to a total antioxidant capacity (CAT) assay using a pro- 319
cedure reported by Prieto et al. (1999) [47]. In a nutshell, 3 ml of reagent solution were 320
combined with 5 µL of each sample. (0.6 M H2SO4, 28 mM Sodium phosphate and 4 mM 321
Ammonium mo-lybdate). For 90 min, the mixes were incubated at 95 °C. following a 20 322
min chilling period at room temperature. At 695 nm, the absorbance of each sample was 323
calculated in comparison to a blank solution. The benchmark is ascorbic acid. Ascorbic 324
acid milligram equivalents per gram of extract (mg EAA/g E) were used to express the 325
results. The experiment was carried out three times. 326
327
2.10. Antimicrobial activity 328
The minimum inhibitory concentration (MIC) of each A. iva extract was determined 329
using a sterile 96-well microtiter plate, employing resazurin as a cell growth indicator [48– 330
51]. A 96-well microtitration plate was made by pouring 100 µL of Mueller Hinton broth 331
for bacteria and the Sabouraud broth for fungi into each well. The first well of the plate 332
was filled with a volume of 100 µL of extract (200 mg/mL) prepared in (30/70) % etha- 333
nol/distilled water, which had been diluted serially by 1/2, 1 /4, 1/8, 1/16, 1/32 and 1/64 334
before being added. Following that, 100 µL of inoculum were added to each well. At 37°C, 335
the microtiter plate were incubated for 24-48 h. Following the addition of 10 µL (6.75 336
mg/mL) of resazurin solution to each well and a 2 h incubation period at 37°C, the MIC of 337
the samples was determined. Reduction in resazurin and subsequent bacterial or fungi 338
growth are shown by a shift from blue to pink. The MIC value was calculated by obtaining 339
the extract at the lowest concentration at which the color change occurred. On brand-new, 340
sterile Mueller-Hinton agar plates and in Sabouraud agar plates, treatment broth from 341
wells with no discernible growth in the MIC assay was cultured to determine the mini- 342
mum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC). 343
These latter were defined as the lowest concentration (maximum dilution) of extract that, 344
after 24 h at 37°C, prevented the growth of colonies on solid agar medium [52]. 345
2.11. Acute toxicity of decoction of Ajuga iva (L.) 346

The objective of the toxicity test was to demonstrate that therapeutic doses of A. iva 347
decoction in normal mice do not cause any short-term harm. In reality, five groups of 348
albino mice (20–35 g) were randomly selected from two batches and divided into five 349
groups (n=6; ♂/♀=1). The treated groups received A. iva decoction doses of 0.5 g/kg, 1 g/kg, 350
or 2 g/kg, while the control group received distilled water (10 ml/kg). The mice were 351
weighed before the test began. They then received an oral dose of the extract immediately 352
after. After that, we kept an eye on them nonstop for 10 h to look for any indications of 353
toxicity.The mice were observed every day for the following 14 days for any new clinical 354
or behavioral indications of harm. This test was carried out in accordance with the recom- 355
mendations of the Organization for Economic Co-operation and Development (OECD) 356
[53]. 357
2.12. Antihyperglycemic effect of decoction of Ajuga Iva (L.) 358

The hypoglycemic effect (post-prandial blood glucose) of A. iva (L.) decoction in vivo 359
was discovered using an oral glucose tolerance test [54]. Three groups of normal rats were 360
formed (n=6; ♂/♀=1): Normal rats were given distilled water (10 ml/kg) as the control 361
group. The treated groups were given either a decoction (400 mg/kg) or glibenclamide (2 362
mg/kg). The oral glucose tolerance test was conducted as follows: Glycaemia was meas- 363
ured at time zero, immediately following the administration of the test substance to the 364
rats. (decoction or glibenclamide). Another blood glucose reading was performed 30 min 365
after the animals had a 2 mg/kg D-glucose excess. The fluctuation in blood glucose was 366
then observed for 2 h. 367
368
2.13. Pancreatic α-amylase inhibitory effect of decoction of Ajuga iva (L.) 369
2.13.1. In-vitro test 370
The method published by Boulfia et al. with minor changes [55] was used to test the 371
decoction's inhibitory effect on the enzymatic activity of pancreatic α -amylase. To 200 µL 372
of the phosphate buffer solution (0.02 M; pH=6.9), different concentrations of the decoc- 373
tion solution were added. The pancreatic α -amylase enzyme solution (61.33 IU) was then 374
diluted to a volume of 200 µL and added to each tube. These underwent a 10 min prein- 375
cubation at 37°C. Then, each tube received 200 µL of the starch solution (0.5 %). For 15 376
min, entire mixture was incubated at 37°C. The enzymatic reaction was stopped by adding 377
600 µL of DNSA (2.5%) to the tubes. After that, the tubes were incubated for 8-min in a 378
bath of boiling water. After applying heat shock to halt the reaction, 10 mL of distilled 379
water was added to each tube and the tubes were placed in an ice bath. A spectrophotom- 380
eter was used to read the absorbance at 540 nm (V-1200). The positive control utilized was 381
acarbose. From the following equation, the % inhibition of each extract and acarbose was 382
determined. The alpha-amylase inhibitory activity was estimated using the following for- 383
mula and reported as a percent inhibition: 384
(A −A )−(A3−A4)
Alpha − amylase inhibitory activity (%) = 100 ∗ [ 1 2 ] (Eq 8) 385
A1−A2
386
𝐴1 : Control absorbance (enzyme + buffer) 387
𝐴2 : Absorbance of the control blank (buffer without enzyme) 388
𝐴3: Sample absorbance (enzyme + extract) 389
𝐴4 : Absorbance of sample blank (extract without enzyme) 390
2.13.2. In-vivo test 391

The purpose of the study was to confirm the decoction inhibiting effect of A. iva (L.), 392
in vivo, in normal rats considering the effect of intestinal lumen on the inhibitory effect of 393
the decoction of A. iva (L.). Three groups of normal rats (180-250 g) were formed after a 14 394
h fast (n=6; ♂/♀=1). The treated groups either received the decocted extract (400 mg/kg) or 395
acarbose (10 mg/kg), whereas the control group received distilled water (10 mL/kg). The 396
oral starch tolerance test was carried out as follows: the product to be tested (distilled 397
water, decoction or Acarbose) was administered (t = 0 min), 30 min later, a blood glucose 398
measurement was carried out, just after; the rats were overloaded with starch (3 g/kg). 399
Then, the variation in blood glucose was measured after 30, 60 and 120 min. 400
2.14. Statistical analysis 401

After undergoing one-way analysis of variance with ANOVA (one-way analysis of 402
variance with Tukey's post hoc test), the data are shown as means and standard deviation. 403
The criteria for statistical significance were P < 0.05, p < 0.01 and p < 0.001. Heat maps and 404
Pearson correlations between the chemical composition of phenolic compounds and their 405
biological activity were produced using the R software. 406
3. Results and discussion 407
3.1. Quality control of Ajuga iva (L.) 408
The quality control of the plant material of A. iva (L.) was carried out, according to 409
the standards cited in supra, by determining the moisture content (MC), the pH, the titrat- 410
able acidity, the ashes and the ICP, the results obtained are shown in Tables 3 and 4 below. 411
3.1.1. Water content 412

The moisture content of the raw powder of A. iva (L.) leaves measured by the oven 413
method is about 11.56 ± 0.164%. This humidity level is linked to the water activity, so the 414
drying of the plants is carried out to ensure good preservation of the samples by inhibiting 415
the enzymatic activity, which prevents the degradation of certain components and the 416
bacterial proliferation [56]. According to the criteria given in the European Pharmacopoeia 417
in 2000, this content must not exceed 10%. The results of this analysis showed that the 418
moisture content of A. iva (L.) was slightly above 10%. Moisture content slightly above 419
10% can improve the medium-term storage of our powder. This water content obtained 420
was considered to be higher than the water content of 6.43% obtained from the leaves of 421
A. remota Bens in Ethiopia [57]. Several factors contribute to this variability, the most im- 422
portant of which are climate, soil, harvest date, and drying and storage methods. 423
3.1.2. pH determination 424

The hydrogen potential tells us about the assimilability and good availability of nu- 425
trients by plants. The measured pH of A. iva (L.) is around 5.28 ± 0.098, this value is in the 426
interval (4.0-6.5), placing our plant in the category of acidophilic plants without total lime- 427
stone. The value found proves that this species has good absorption property of nutrients 428
like iron, which is well confirmed by the results of ICP analysis. 429
3.1.3. Titratable acidity 430

The titratable acidity value found in A. iva (L.) powder is equal to (37.866 ± 0.0075) % 431
(Table 3). The titratable acidity indicates the quantity of free organic anions in the solution 432
tested. According to Soro and coworkers, pH is inversely proportional to titratable acidity 433
[58]. 434
3.1.4. Ash content 435

Ashes are a sort of mineral compound that, unlike organic materials, do not become 436
volatile at high temperatures, hence their percentage tells us about the amount of these 437
compounds. A. iva (L.) records a high ash content of around (16.3005 ± 0.2763) %. The rate 438
recorded is slightly higher than those obtained at A. iva (L.) in Tunisia (13.0%) [59] and in 439
Algeria (11.10%) [60]. Also, this rate is much lower than that found in A. remota Benth from 440
Ethiopia (27.10%) [57]. According to Ammar and his collaborators [61], the ash content of 441
the leaves of A. iva (L.) varies from one region to another, indeed, they found, for the two 442
localities of Tunisia; Mograne and Nabeul, contents varying between 15.2% and 24.3%. 443
These differences between localities could be due to some external factors such as climatic, 444
soil and environmental conditions. The content found makes our plant an important 445
source of minerals for human nutrition. 446
Table 3. Parameters evaluated of plant material from Ajuga iva (L.). 447
Plant Moisture content (%) pH Ash (%) Titratable acidity (%)

A. iva 11.56 ± 0.16 5.28 ± 0.10 16.30 ± 0.28 37.87± 0.01
448
3.2. Quantification of metals 449

Determination of the content of minerals, including microelements iron (Fe), copper 450
(Cu), and heavy metals cadmium (Cd), lead (Pb), antimony (Sb), chromium (Cr), arsenic 451
(As), and titanium (Ti) was carried out in the leaves of A. iva (L.), the results of which are 452
presented in Table 4. The results obtained indicate that iron was the most abundant mi- 453
croelement with a content of 2.3433 mg/g. For copper, a low concentration was noticed in 454
the order of 0.0082 mg/g. As for heavy metals, considerable levels were noted as follows: 455
Cr (0.0297)Pb (0.0243) Sb (0.0239) As (0.0213) Cd (0.0015). 456
Thus, the leaves of A. iva (L.) are characterized by their richness in minerals, which 457
may be responsible for their biological activity. Senhaji Souad and co-workers [62] re- 458
ported that the metal analysis result in A. iva (L.). pseudova (DC.) Briq collected in the re- 459
gion of Taza (Morocco) showed an abundance of iron in comparison with the other ele- 460
ments measured. This comparison of results agrees with ours, where we found the Iron 461
concentration to be the highest in our sample. 462
463
464
465
Table 4: Heavy metal content (mg/g) (ICP) of plant material from Ajuga iva (L.). 466
Ajuga iva (L.) As Cr Sb Pb Cd Fe Cu Ti

Content (mg/g) 0.0213 0.0297 0.0239 0.0243 0.0015 2.3433 0.0082 0.0257
467
Note that iron (Fe) is a necessary element for both humans and animals, as well as a 468
necessary part of myoglobin and hemoglobin. Anemia can result from iron deficiency, 469
which also causes gastrointestinal infections, nosebleeds, and cardiac infarctions. Reactive 470
oxygen species, which Fe can produce, play a role in the pathophysiology of diabetes and 471
its side effects such as diabetic nephropathy. Thus, the use of a decoction of Ajuga iva (L.), 472
provides a value of 234.33 mg of iron per 100 g of plant, which is pharmacologically sig- 473
nificant in the absence of dietary iron [63,64]. 474
For Copper (Cu), it is a component of several enzymes that plays a role in the syn- 475
thesis of collagen and the regulation of cardiovascular function. However, its severe and 476
chronic deficiency promotes anemia, causes cardiovascular changes and skeletal weak- 477
ness. The estimated dietary and adequate intake for Cu2+ is only 0.9 mg. Our results record 478
a content of 0.82 mg/100 g of Ajuga iva (L.), which is physiologically useful [63]. 479
Lead (Pb) is a chemical that is frequently dangerous and is known to have a variety 480
of toxic effects in living things, including those that are morphological, physiological, and 481
biochemical in nature [65,66]. The temporary tolerable weekly intake for adults has been 482
set by the FAO/WHO committee at 3 mg per person. Our findings revealed a physiologi- 483
cally acceptable Pb concentration of 2.43 mg/100 g of Ajuga iva [67]. 484
Chromium (Cr) is an essential mineral element in minute amounts in humans for 485
sugar and lipid metabolism, used as a glucose tolerance factor (GTF), and its deficiency 486
can cause a disease called Cr deficiency, while its hexavalent form is extremely toxic and 487
carcinogenic. In fact, the Recommended Dietary Allowance (RDA) reported in a review, 488
that people with diabetes when given a dose of 150-250 µg/day allowed them to correct 489
their diabetes symptoms. Thus, chromium supplements can help treat glucose intolerance 490
and type 2 diabetes [63,64]. It should be noted that 5.22 g of A. iva (L.) can yield 155 µg/d 491
of Cr; this quantity is adequate for diabetic people. As a result, this plant can be thought 492
of as a successful treatment for type 2 diabetes and other conditions brought on by chro- 493
mium deficiency). 494
Cadmium (Cd) is a non-essential metal mainly stored in the liver and kidneys, it in- 495
duces renal, cerebral, cardiac dysfunction and lung and reproductive disorders. It has 496
been officially listed as a rat and human lung carcinogen by the International Agency for 497
Research on Cancer [64–68]. The FAO/WHO committee has proposed adopting a tempo- 498
rary tolerable weekly intake of 400 to 500 µg per person [67]. It should be mentioned that 499
taking 450 µg weekly of Cd, obtained from 300 g of A. iva (L.) is within the limits of the 500
value prescribed by FAO/WHO. 501
Arsenic (As) is a poisonous metalloid and one of the most significant environmental 502
contaminants in the world. Inorganic arsenic is particularly harmful because it can have 503
both short-term and long-term negative health consequences. According to recent reports, 504
arsenic is a common environmental carcinogen that has been related to diabetes, cardio- 505
vascular disease, skin cancer, bladder cancer, and when exposed to it. The results obtained 506
on A. iva (L.) recorded a content of As approximately 21.3 µg/g, which is within the range 507
authorized for arsenic 1-30 µg/g by FAO/WHO [64,69–71]. 508
Antimony (Sb) is a very rare element, often found in sulphides and sulfur salts. It 509
exhibits a biochemical behavior comparable to that of arsenic and bismuth. Antimony's 510
toxicity is poorly understood, but Sb (III) species are often more toxic than Sb (V) species 511
[72,73]. In a review prepared by Boland and collaborators, they reported that plants could 512
be good potential phytoremediators for the remediation of metals, including Sb, and that 513
the appropriate concentration of Sb in plant pytoremediators ranged from 3.92 to 143.69 514
mg/kg [74]. Our results recorded a concentration of the order of 23.9 mg/kg which is in- 515
cluded in the range of suitable and non-toxic concentrations. 516
3.3. Phytochemical screening 517

The primary and secondary metabolites detected in A. iva (L.) leaves extracts are 518
shown in Table 5. The results of these tests show that A. iva (L.) leaves are rich in primary 519
metabolites. Indeed, A. iva (L.) contains a large amount of lipids followed by proteins and 520
traces of reducing sugars and glycogen. With regard to secondary metabolites, A. iva (L.) 521
is characterized by the presence of reducing compounds, oses and holosides in large quan- 522
tities. Also, gallic and catechin tannins, flavones and leucoanthocyanins, anthocyanins, 523
sterols and triterpenes are present in the leaves of this plant. Alkaloids are present in the 524
form of traces, while mucilages and saponosides are totally absent in the leaves of this 525
plant. A study by Senhaji and his collaborators also showed that A. iva (L.) is rich in cate- 526
chic tannins, saponins, sterols, and flavonoids[62]. 527
Table 5. Phytochemical screening of Ajuga iva (L.) leaves. 528
Chemical groups Observations

Polysaccharides + Glycogen
Lipids +++
Primary metabolites R.Biuret +
Proteins
R.Xantho ++
Reducing sugars +
Gallic ++
Tannins
catechists ++
Flavones ++
Flavonoids
Leucoanthocyanins ++
Anthocyanins ++
Saponosides -
Secondary metabolites Mayer +
Alkaloids Dragendorff +
Wagner ++
Reducing compounds +++
Monosaccharides and holosides +++
Mucilages -
Sterols and triterpenes ++
+: Weak positive test; ++: Positive test; +++: Strongly positive test; −: Negative test. 529
3.4. Determination of extraction yields and contents of total polyphenols, flavonoids and 530
catechin tannins 531
The results of the extraction yield and the contents of polyphenols, flavonoids and 532
catechin tannins of A. iva (L.) according to the extraction methods used, are indicated in 533
Table 6. We find that the extraction by decoction gives the greatest yield (17.01 6 ± 0.032) 534
%, compared to the extraction by Soxhlet whose aqueous extract has the value (11.048 ± 535
0.511) % while the hydroethanolic extract records the value of (7.385 ± 0.662) %. These 536
results agree with several similar studies, which showed that aqueous extracts showed 537
higher yields than extracts obtained by organic solvents [75–77]. For example, extraction 538
yields determined from extracts of A. iva (L.) obtained by acetone, ethanol and water were 539
respectively around 2.3%, 2.6% and 24% [18]. Also, the extraction yield seems to be influ- 540
enced by the degree of polarity of the solvent, the extraction method, the extraction time 541
as well as by the degree of polarity of the various components of the extract such as the 542
phenolic constituents [33]. With regard to the polyphenol and flavonoid contents, we note 543
that the hydro-ethanolic extract presents (69,850 ± 2,783) mg EAG/gE; (17.127 ± 0.474) mg 544
EQ/gE respectively, these values are much higher than those evaluated in the case of the 545
aqueous extract (53.148 ± 2.509) mg EAG/g E; (10.281 ± 0.521) mg EQ/gE, respectively and 546
the discount (40.786 ± 2.957) mg EAG/g E; (8.960 ± 0.370) mg EQ/g DE, respectively. As for 547
the catechic tannin content, the three extracts (decocted, hydro-ethanolic and aqueous) 548
seem to have similar quantities, respectively: (5.746 ± 0.413) mg EQC/gE; (5.566 ± 0.000) 549
mg EQC/gE; (5.792 ± 0.064) mg EQC/gE. These results agree with a study carried out by 550
Saad and his collaborators[78] , they found that the methanolic extract is richer in poly- 551
phenols and flavonoids compared to the aqueous extract. This difference could be justified 552
by the degree of polarity of each solvent to drive the phenolic compounds [78]. These 553
phenolic compounds are secondary metabolites that have been extensively researched in 554
a variety of medicinal plants, fruits, and vegetables [79,80]. They have antioxidant, anti- 555
bacterial, and carbohydrase inhibitor properties. Additionally, flavonoids are a subclass 556
of polyphenolic chemicals with a benzo-γ-pyrone structure that have shown to be effec- 557
tive against microorganisms[81]. 558
Table 6. Extraction yields and phenolic compound contents of Ajuga iva (L.) extracts. 559
Polyphenol Flavonoid Catechic tannin

Extract Extraction yields (%)
(mg EAG/g DE) (mg EQ/g DE) (mg EQC/g DE)
Decocted 17.016 ± 0.032 40.786 ± 2.957 8.960 ± 0.370 5.746 ± 0.413
Aqueous extract 11.048 ± 0.511 53.148 ± 2.509 10.281 ± 0.521 5.792 ± 0.064
Hydroethanolic extract 7.385 ± 0.662 69.850± 2.783 17.127± 0.474 5.566 ± 0.000
3.5. Identification of the chemical composition of phenolic compounds from the decoction of 560
Ajuga iva (L.) by LC/UV/ESI-MS 561
The analysis of the chemical composition of the polyphenols from the leaves of 562
A. iva (L.) was carried out by LC/UV/ESI-MS. The chromatographic profile presented be- 563
low (Figure 2) illustrates the peaks of the constituents with their retention times and their 564
relative abundance (%). Based on analytical and spectroscopic data (chromatogram and 565
mass spectra), we detected and identified 37 compounds whose names are presented in 566
Table 7. All compounds were identified by interpreting their determined mass spectra 567
and considering data reported in the literature. We have identified ten major compounds 568
whose relative abundance is greater than 3%, these are ferulic acid (19.06%), quercetin 569
(10.19%), coumaric acid (9.63%), apigenin -7-(2-O-apiosylglucoside) (6.8%), cholesterol 570
(6.17%), luteolin (4.53%), ajugasterone D (4.29%), kaempferide (4.2%), epigallocatechin 571
gallate (3.94%) and vanillin (3.17%). ferulic acid, quercetin and coumaric acid were the 572
most abundant compounds found in the decoction of A. iva (L.). Various studies have 573
shown the pharmacological properties of ferulic acid as an antioxidant [82,83], antialler- 574
gic, anti-inflammatory, hepatoprotective, anticancer, antimicrobial, antiviral, vasodilator, 575
antithrombotic [84] and antidiabetic [85]. The same goes for the compound quercetin 576
which is known to have good anti-inflammatory, antioxidant [86], antidiabetic [87], anti- 577
cancer, antiproliferative, apoptotic [88,89], antimicrobial and antidiabetic effects [90]. In 578
addition, coumaric acid also has antimicrobial, antioxidant, anticancer, immunoregula- 579
tory [91,92], antidiabetic and antihyperlipidemic properties [93]. Moreover, new com- 580
pounds called apigénine-7-(2-O-apiosylglucoside), ajugastérone D, and cholestérole have 581
been discovered as a result of our research on the decoction of A. iva (L.). Since ferulic acid 582
is a derivative of cinnamic acid, many studies on the chemical composition of the genus 583
Ajuga have been indicated the presence of this bioactive compound and the existence of 584
p-coumaric acid, quercetin, and luteolin [94–96]. These results in agreement with those of 585
our study. Other chemical components found in our plant, such as: epigallocatechin gal- 586
late, harpagide, and cyasterone, have also been reported in A. iva (L.) from Tunisia [61]. 587
In conclusion, these results can confirm the use of A. iva (L.) leaves as a natural alternative 588
to industrial products, especially in the pharmaceutical field. 589
Figure 2. HPLC-MS chromatogram detected phenolic compounds from Ajuga iva (L.) decoction. 590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
Table 7. Chemical composition of Ajuga iva (L.) decoction by LC/UV/MS. 609
[M-H] - [M+H] +
N° TR (min) A.R. (%) MW Compounds identified
(m/z) (m/z)
1 3.12 0.43 195 - 196 Galacturonic acid
2 3.9 0.79 317 - 318 Myricetin
3 4.14 0.73 169 - 170 Gallic acid
4 4.84 1.37 175 - 176 Ascorbic acid
5 5.56 1.3 209 - 210 Mucic acid
6 5.82 0.33 173 - 174 Arginine
7 6.18 3.17 151 - 152 Vanillin
8 7.53 2.01 191 - 192 Quinic acid
9 8.78 1.06 147 - 148 Cinnamic acid
10 12.67 1.3 315 - 316 Rhamnetin
11 13.07 2.04 451 - 452 Catechin-7-O-glucoside
12 14.35 2.43 289 - 290 Catechin
13 15.34 1.39 363 - 364 Harpagid
14 16.77 4.29 477 - 478 Ajugasterone D
15 17.33 1.41 145 - 146 Coumarin
16 17.99 1.26 - 357 356 Ferulic acid 4-O-glucoside
17 18.43 9.63 163 - 164 Coumaric acid
18 19.4 3.94 - 459 458 Epigallocatechin gallate
19 20.73 1.96 405 - 406 8-O-acetyl-harpagid
20 21.12 19.06 193 - 194 Ferulic acid
21 21.56 6.17 385 - 386 Cholesterol
22 21.9 0.77 519 - 520 Cyasterone
23 22.72 1.88 389 - 390 Resveratrol 3-Glucoside
24 23.75 6.8 563 - 564 Apigenin-7-(2-O-apiosylglucoside)
25 24.03 0.43 - 651 650 Apigenin 7-O-(6''-malonyl-apiosyl-glucoside)
26 24.17 1.38 269 - 270 Apigenin
27 24.65 1.45 565 - 566 Quercetin-3-O-pentosyl-pentoside
28 25.02 10.19 301 - 302 Quercetin
29 25.12 4.25 299 - 300 Kaempferide
30 25.35 4.53 387 - 388 Luteolin
31 25.61 1.87 - 327 326 Trans-p-coumaric acid
32 26.49 0.31 329 - 330 Vanillic acid glucoside
Total: 99.93 %
610
3.6. Antioxidant activity of different Ajuga iva (L.) extracts 611

The antioxidant activity of different A. iva (L.) extracts is determined using three tests, 612
DPPH, CAT, and FRAP. This antioxidant property for each extract is estimated by deter- 613
mining the 50% inhibitory concentration (IC 50). The results of the antioxidant activity of 614
A. iva (L.) depending on the extraction method used are shown in Table 8. The hydro- 615
ethanolic extract showed the highest ability to scavenge free radicals of DPPH* (IC 50 =59.92 616
± 0.70 µg/mL), followed by the aqueous extract prepared by Soxhlet (IC 50=145.15 ± 0.72 617
µg/mL), then the decoction which showed the lowest antioxidant activity (IC50=195.35 ± 618
14.58 µg /mL). The antioxidant activity of the extracts of this plant was also evaluated by 619
testing their iron-reducing antioxidant power. Similarly, the hydro-ethanolic extract 620
showed the highest reducing power (EC50=196.85 ± 1.54 µg/mL), followed by the aqueous 621
extract prepared by soxhlet (EC50=290.04 ± 0.06 µg/mL), then the decocted which showed 622
the lowest reducing power (EC50=346.79 ± 3.59 µg/mL). For the total antioxidant capacity 623
test, the hydro-ethanolic extract showed the highest total antioxidant capacity (199.21 ± 624
0.37 mg EAG/gE), followed by the aqueous extract prepared by Soxhlet (109.55 ± 2.24 mg 625
EAG /gE), then the decoction showed the lowest total antioxidant capacity (117.33 ± 0.69 626
mg EAG/gE). The antioxidant activity of all these extracts remains lower in comparison 627
with that of ascorbic acid used as a positive control (Table 8). The antioxidant activity of 628
A. iva (L.) in this study is higher than the antioxidant activity of the same plant in other 629
regions like A. iva (L.) from Oued Amlil region, Taza, Morocco [78], from the region from 630
Bel-Abbass in northwestern Algeria [33], and A. chamaecistus from Taleqan region, Alborz 631
province, Iran [97], using the DPPH test. This very significant antioxidant property of A. 632
iva (L.) extracts is due to their richness in polyphenols and flavonoids. Indeed, several 633
studies have reported that these secondary metabolites have very significant antioxidant 634
activity [98,99]. Indeed, Makni and his collaborators worked on several extracts of A. iva 635
(L.) using four solvents (Methanol, chloroform, Water and Hexane) and found that the 636
methanolic and aqueous extracts having the highest contents of phenolic compounds and 637
flavonoids revealed the strongest antioxidant powers for all tests (DPPH*, FRAP and 638
CAT) [100]. This difference in the antioxidant activity of the three extracts studied is also 639
mentioned in the work of Saad et al., (2019) where they showed that the methanolic extract 640
of A. iva (L.) has the highest antioxidant capacity compared to its aqueous extract [78]. 641
Table 8. Antioxidant activities of Ajuga iva (L.) extracts (by different methods). 642
DPPH* CAT FRAP

Extracts
IC50 (µg/mL) (mg EAG/gE) EC50 (µg/mL)
Decocted 195.35 ± 14.58 117.33 ± 0.69 346.79 ± 3.59
Aqueous extract 145.15 ± 0.72 109.55 ± 2.24 290.04 ± 0.06
Hydro-ethanolic extract 59.92 ± 0.70 199.21 ± 0.37 196.85 ± 1.54
Ascorbic acid 3.23 ± 0.11 - 9.27± 0.00
3.7. Correlations between antioxidant activities and phenolic compound contents of Ajuga iva 643
(L.) extracts 644
The study of the correlation between the contents of phenolic compounds and the 645
antioxidant activities of A. iva (L.) extracts was established by determining the linear cor- 646
relation coefficient (R2), known as the Bravais-Pearson coefficient. The results grouped 647
together in Figure 3 show the correlation between the antioxidant activities (DPPH*, FRAP 648
and CAT) and the contents of phenolic compounds (polyphenols, flavonoids and tannins). 649
Poly
650
1
651
Poly 1,0 Flav
0,8
652
0,6
653
Flav 0,96 1,0 TC
0,4
654
TC 655 -0,81 -0,94 1,0 CAT 0,2
656 0
CAT
657 0,87 0,97 -0,99 1,0 DPPH -0,2
658 -0,4
DPPH -1,00 -0,97 0,84 -0,90 1,0 FRAP
659 -0,6
660
-0,8
FRAP -1,00 -0,97 0,84 -0,89 1,00 1,0
661
-1
662
663
Figure 3. Correlation coefficient between the variables of polyphenolic compounds and biological 664
activities in the extracts of the leaves of Ajuga iva (L.) from the region of Masmouda-Morocco. 665
666
We notice a strongly negative correlation between the contents of polyphenols, fla- 667
vonoids and the antioxidant activities DPPH* and FRAP which gave coefficients of the 668
order of -1 and -0.97 respectively. In addition, a strong positive correlation was observed 669
between polyphenols, flavonoids and total antioxidant activity with respective correlation 670
coefficients: R2=0.87 and R2=0.97. On the other hand, the tannins show no correlation with 671
the antioxidant activities FRAP, DPPH* and CAT, the correlation coefficients of which are 672
as follows (R2FRAP/Tanins=0.84, R2DPPH/Tanins=0.84 and R2CAT/Tanins= -0.99). From this, we deduced 673
that the antioxidant power of the DPPH*, CAT and FRAP tests of the A. iva (L.) extracts 674
was due to the contribution of more than 87% of phenolic compounds and flavonoids, 675
while the tannin content was very weak. In addition, a significant positive association be- 676
tween polyphenols and flavonoids (R2=0.98) the opposite hand, the non-correlation was 677
observed between catechin tannins with polyphenols and flavonoids. However, there is a 678
strong positive correlation between the reducing power of iron (R2 = 1) and the DPPH* 679
radical scavenging ability. 680
The antioxidant activities of DPPH* and FRAP have a high negative correlation with 681
the total antioxidant activity. R2=-0.90 and R2=-0.89, respectively, are the negative correla- 682
tion coefficients of correlation. This indicates that in the extracts tested, the free radical 683
scavenging power (DPPH*) and the iron reducing power (FRAP) are inversely propor- 684
tional to the total antioxidant capacity. These results are in accordance with the data from 685
the literature, of which several authors have shown the dependence between the potential 686
antioxidant activities of an extract and the content of phenolic compound [101–103]. 687
3.8. Antimicrobial Activity of Ajuga iva Extracts 688
3.8.1. Antifungal activity 689

The antifungal activity of the three extracts (Eeth, EAq and Ed) of Ajuga iva (L.) was 690
evaluated against eight fungal strains C. albicans, C. dubliniensis, S. cerevisiae, A. niger, C. 691
tropicalis, C. krusei, and C. glabrata, and these strains sensitive to the extracts were tested 692
by the MIC and MBC methods, the results are depicted in Table 9. 693
Table 9. Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MBC) 694
of Ajuga iva (L.) extracts (mg/mL). 695
MIC Extracts (mg/mL)

Microorganism
Decocted Aqueous Hydroethanolic
MIC MBC MIC MBC MIC MBC
Candida albicans 12.5 50.0 25.0 25.0 12.5 25.0
Candida dubliniensis 25.0 25.0 50.0 50.0 25.0 25.0
Saccharomyces cerevisiae 12.5 25.0 25.0 25.0 50.0 50.0
Aspergillus niger 50.0 50.0 25.0 25.0 25.0 25.0
Candida tropicalis 50.0 50.0 25.0 25.0 50.0 50.0
Candida krusei 12.5 25.0 25.0 25.0 50.0 100.0
Candida glabrata 12.5 25.0 25.0 25.0 25.0 50.0
Candida parapsilosis 6.25 12.5 6.25 12.5 6.25 12.5
696
The determination of the MIC of the extracts, with a range of concentration going 697
from 12.5 to 100 mg/mL was not influenced neither by the type of extraction (Soxhlet or 698
decoction) nor by the type of solvent (water and the mixture ethanol-water), the antifungal 699
activity depended on the fungal species; the results obtained showed that the C. parapsilo- 700
sis strain is the most sensitive to the three extracts of A. iva (L.), the MIC appears from the 701
concentration of 6.25 mg/mL. The MICs recorded for the other strains varied depending 702
on the type of extract, ranging from 6.25 mg/mL to 100 mg/mL. Indeed, C. dubliniensis, C. 703
glabrata and A. niger resisted up to 25 mg/ mL for the hydroethanolic extract whereas the 704
strains C. albicans, S. cerevisiae, A. niger, C. tropicalis, C. krusei, C. glabrata resisted up to 25 705
mg/ mL for the aqueous extract. Furthermore C. albicans S. cerevisiae, C. krusei, C. glabrata 706
resisted up to 12.5 mg/ mL for the decocted extract. The MBCs differ according to the fungal 707
species tested. C. parapsilosis was the most sensitive towards hydroethanolic, aqueous and 708
decocted extracts with a concentration of 6.25 mg/ml (Table 9). 709
A. iva (L.) has proven to have antibacterial characteristics in a number of investiga- 710
tions [30,33,100,104–106]. The antimicrobial study on A. iva (L.) Tunisien demonstrated 711
that methanolic extracts displayed promising antibacterial and antifungal activities[100]. 712
This study tested three different fungal strains, A. clavatus, A. niger, and Fusarium, with 713
Fusarium exhibiting the highest inhibitory activity and A. niger exhibiting the lowest in- 714
hibitory activity. The findings provide evidence that phenolic compounds of A. iva may 715
be responsible for the antifungal/antimicrobial activities [107,108]. 716
3.8.2. Antibacterial activity 717

According to the results of the antibacterial activity (Table 10), the MIC of the aqueous 718
extract was revealed from a concentration of 3.13 mg/ml for P. mirabilis up to 100 mg/mL 719
for S. epidermidis and E. coli BLSE for aqueous extract, while hydroethanolic extract recorded 720
MICs ranging from 12.25 mg/mL for S. agalactiae to 50 mg/mL for S epidermidis and P. ae- 721
ruginosa. Similarly, the decoction was more effective against P. mirabilis with a concentra- 722
tion of 12.5 mg/mL against the strains P. mirabilis and S. aureus BLACT to 100mg/mL for E. 723
coli BLSE. The lowest MBC values were recorded for P. mirabilis (12.5 mg/mL), while those 724
highest for E. coli ESBL (100mg/mL). We can then say that P. mirabilis was the most sensi- 725
tive, while E. coli ESBL was the most resistant. According to a study, the antibacterial po- 726
tential of hydroalcoholic and aqueous extracts by the MIC method was qualitatively eval- 727
uated against four Gram-positive strains and three Gram-negative strains. In fact, the 728
aqueous extract showed to be slightly more effective than the extract hydroalcoholic 729
against the growth of all tested bacteria. A significant amount of activity was shown 730
against the S. aureus (MRSA) strain (80 < MIC < 155 µg/disk), and a considerable amount 731
of activity was shown against the S. aureus and B. cereus strains (145 < MIC < 200 µg/disk). 732
L. monocytogenes and all Gram-negative bacteria, on the other hand, were only faintly in- 733
hibited (625 < MIC < 1250 µg/disc) [33]. The results obtained demonstrate that there are 734
differences between the effect of the extracts studied and the bacterial species. The syner- 735
gistic interaction of all the chemical elements found in the extracts is what gives A. iva (L.) 736
its strong antibacterial activity (Table 10). Characterized flavonoids may be the cause of 737
A. iva's powerful antibacterial activity. Different levels of these substances' effects on mi- 738
crobial development are possible. Some flavonoids combine with extracellular proteins to 739
create complexes and are thus only soluble with bacterial cell proteins [109]. These com- 740
plexes affect the permeability of microbial membranes [110] and disrupt the structure of 741
their various layers of polysaccharides, fatty acids and phospholipids [110]. The promis- 742
ing antibacterial and antifungal properties of A. iva (L.) according to our study, have 743
demonstrated that certain compounds may be further evaluated as antimicrobial medi- 744
cines for commercialization. 745
746
747
748
749
750
751
752
753
754
Table 10. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration 755
(MBC) of Ajuga iva (L.) extracts (mg/ml). 756
Bacteria Extracts (mg/mL)

Decocted Aqueous extract Hydroethanolic

MIC MBC MIC MBC MIC MBC
Staphylococcus epidermidis 50.0 100.0 100.0 50.0 50.0 100.0
Staphylococcus aureus BLACT 12.25 50.0 12.25 50.0 25.0 25.0
Streptococcus agalactiae (B) 50.0 100.0 25.0 25.0 12.25 25.0
Escherichia coli sauvage 50.0 50.0 25.0 25.0 25.0 25.0
Escherichia coli BLSE 100.0 100.0 100.0 100.0 25.0 50.0
Entérobactérie cloacae 50.0 100.0 25.0 25.0 25.0 25.0
Klebsiella pneumoniae 50.0 50.0 25.0 50.0 25.0 50.0
Proteus mirabilis 12.5 25.0 3.13 12.5 25.0 25.0
Pseudomonas aeruginosa 50.0 50.0 50.0 50.0 50.0 50.0
757
3.9. Acute safety of the decocted extract of Ajuga iva (L.) 758
The decoction of this plant was not toxic even at 2 g/kg, according to the results of 759
the acute toxicity test. Throughout the entire follow-up period, it did not result in any 760
toxicity symptoms (such as diarrhea, vomiting, altered movement, etc.) or fatalities. These 761
results agree completely with that found by Saad et al [78]. They confirmed that the meth- 762
anolic and aqueous extract had no short-term adverse effects in mice even at 2 g/kg [78]. 763
The consumption of ivette by normal people; does not lead to a reduction in their blood 764
sugar levels, whereas it has a hypoglycemic effect in people with diabetes [22]. By the oral 765
route, the LD50 is greater than 14g/kg BW while that of the intraperitoneal route is approx- 766
imately 3.6 g/kg BW. As for the chronic treatment (up to 600 mg/Kg BW), it does not cause 767
any harmful effect on the biochemical and hematological parameters. Histological analy- 768
sis of vital organs reflects normal anatomorphological structures [111], the LD50 shows 769
that the flavonoids of A. iva (L.) are not toxic due to their high values (3600 mg/kg for mice 770
and 4800 mg/kg for rats) [112]. 771
3.10. Antihyperglycemic effect of decocted extract of Ajuga iva (L.) 772

The antihyperglycemic effect is tested for the decoction of A. iva (L.) leaves. The 773
choice of plant material and extraction technique among the other two mentioned above 774
is based on the traditional use of this plant in traditional medicine [113]. Oral administra- 775
tion of an aqueous extract of A. iva (L.) at 400 mg/Kg, 30 min before glucose overload in 776
normal rats, made it possible to significantly reduce postprandial hyperglycemia at 60 777
min (p<0.001, 0.94± 0.22 g/L), and at 90 min (p<0.01, 0.82 ± 0.16 g/L). In a similar manner, 778
glibenclamide significantly reduced postprandial hyperglycemia in the 2 h that followed 779
glucose overload at 60 min (p<0.001; 1.08 ± 0.09 g/L), and at 90 min (p<0.05; 1.09 ± 0.10g/L). 780
At 150 min, no significant difference in glycaemia was observed between all the groups, 781
compared to the group of rats pretreated with distilled water (at 60 min (1.37 ± 0.16 g/L), 782
at 90 min (1.22 ± 0.11 g/L) and at 150 min (0.76 ± 0.07 g/L)) (Figure 4). The area under the 783
curve (AUC glucose) was also substantially lower (p<0.01) in rats administered with the 784
aqueous extract of A. iva (L.) (49.81 ± 7.61 g/L/h) than in rats treated with distilled water 785
(62.91 ± 4.32 g/L/h). Additionally, glibenclamide's area under the curve was considerably 786
(p<0.01) lower (55.95 ± 1.69 g/L/h) than the area under the curve for rats receiving distilled 787
water (62.91 ± 4.32 g/L/hr) (Figure 4). According to our knowledge, many studies have 788
generally assessed the hypoglycemic effect of this plant in different animal models of di- 789
abetes [28,78,113–115], while few studies that have been interested in the mechanism of 790
action of this study. Therefore, in this study we tried to test whether this already proven 791
anti-diabetic effect could be due to its postprandial anti-hyperglycemic ability, using the 792
oral glucose tolerance test. As we mentioned in the results of this test, the antidiabetic 793
effect of this plant is due to its property of reducing the increase in blood sugar caused by 794
the overload of foods rich in carbohydrates. Phytochemical analysis of the aqueous extract 795
of the aerial part of A. iva (L.) showed its richness in apigenin (flavonoid compound) [116], 796
this flavonoid molecule showed a hypoglycemic effect in diabetic rats by reducing glucose 797
absorption, stimulating insulin secretion and stimulating glycogen synthesis in the mus- 798
cles. This finding implies that this flavonoid may have two opposing effects by promoting 799
both glycogen production and insulin secretion (antihyperglycemic) (insulin mimetic) 800
[117–119]. Also, a phytochemical analysis of the aerial section of Ajuga iva found that an 801
essential steroid component called ecdysterone was present in the aqueous extract. This 802
compound has the ability to potentiate the utilization of glucose by cells [120]. In fact, 803
regardless of the concentration of insulin present and without causing any adverse effects, 804
this steroid had this impact on hepatocytes [121]. 805
806
807
808
809
810
811
812
813
814
815
816
817
818
819
Figure 4. variation in 820

postprandial glycaemia (A) and in the area of the postprandial curve (B) in normal rats after admin- 821
istration of the products tested (decoction and glibenclamide at 60 min). Values are SEM means. 822
(n=6). **p<0.01; * p < 0.05: compared to the control. 823
3.11. Inhibitory effect of decocted extract of Ajuga iva (L.) on pancreatic α-amylase 824
3.11.1. In-vitro test 825

Glucose is produced during the catabolism of complex carbohydrates such as starch, 826
and its absorption raises postprandial glycaemia. Therefore, inhibition of enzymes that 827
mediate the intestinal digestion of these carbohydrates is an important strategy for con- 828
trolling blood sugar in diabetics [113]. Acarbose is a drug that inhibits the enzymatic ac- 829
tivity of the enzymes of the brush border, maltase and sucrase, glucoamylase, dextrinase, 830
as well as pancreatic α-amylase [122]. Despite the use of this molecule as an antidiabetic 831
agent, taking refuge an alternative, plant-based antidiabetic product is highly essential. 832
Additionally, long-term consumption of acarbose has caused side effects and rare cases of 833
hepatotoxicity [123]. This is why scientists are interested in functional foods and nutri- 834
tional therapies with curative and preventive effects against diabetes and obesity [124]. 835
The results of the effect of A. iva (L.) decoction on pancreatic α-amylase activity, in vitro, 836
are shown in Figure 5. Indeed, pancreatic α-amylase activity was significantly inhibited 837
in the presence of the aqueous extract with an IC 50 of 1.52 ± 0.03 mg/mL. Moreover, this 838
inhibitory property of the aqueous extract on the enzymatic activity of pancreatic α-am- 839
ylase was superior to that of acarbose (IC 50 = 0.53 ± 0.01 mg/mL). As far as we are aware, 840
not many studies have looked into how this herb affects pancreatic α-amylase. In fact, the 841
inhibitory effect of our aqueous extract on pancreatic α -amylase activity is substantially 842
lower than that seen by Fettach and colleagues for an aqueous extract of the same plant in 843
the region of Oued Amlil, Taza, Morocco (IC50 = 0.21± 0.00 mg/mL) [78], and it is higher 844
than the value found by Senhaji and his collaborators for the same plant in the Taza region 845
(2.21 ± 0.24 mg/mL). 846
847
848
849
850
851
852
853
854
855
856
Figure 5: Inhibitory effect on α-amylase activity by aqueous extract of Ajuga iva (L.) and 857
acarbose, in vitro. Values are means ± SEM, (n=3). 858
3.11.2. In vivo test 859
This test was validated in vivo using the Bouhrim and al. [125] approach to account 860
for the impact of the intestinal environment on the inhibitory action of this plant on the α- 861
amylase enzyme. And according to our knowledge, no studies have been performed re- 862
garding the inhibitory effect of A. iva (L.) decoction on α-amylase, in vivo. For this reason, 863
the oral administration of A. iva (L.) decoction at 400 mg/kg, 30 min before the starch over- 864
load in normal rats allowed for a significant reduction in postprandial hyperglycemia at 865
60 min (p<0.001, 0.84 ± 0.05 g/L), at 90 min (p<0.001, 0.85 ± 0.03 g/L) and at 120 min (p<0.001; 866
0.86 ± 0.06) compared to the group of rats pretreated with distilled water, one of which 867
starch overload was induced with remarkable hyperglycemia at 60 min (1.07 ± 0.02 g/L), 868
90 min (1.15 ± 0.07 g/L) and 120 min (1.05 ± 0.04 g/L). In comparison to the group of rats 869
pretreated with distilled water, acarbose significantly reduced postprandial hyperglyce- 870
mia at 60 min 60 min (p<0.001, 0.89 ± 0.06 g/L), at 90 min (p<0.001, 0.85 ± 0.08 g/L) and at 871
120 min (p<0.001, 0.78 ± 0.09 g/L) over the 2 h after starch excess. (Figure. 6A). Additionally, 872
rats fed with the decoction had an area under the curve (AUC glucose) that was consider- 873
ably lower (p<0.001) than rats treated with distilled water (61.82 ± 1.53 g/L/h). Addition- 874
ally, the area under the curve for acarbose was substantially (p<0.001) lower (52.05 ± 4.27 875
g/L/h) than it was for rats administered with water (61.82 ± 1.53 g/L/h) (Figure 6B). The 876
inhibitory effectiveness of this plant extract in comparison to acarbose is confirmed by this 877
in vivo test. 878
879
880
881
882
883
884
885
886
Figure 6. Effect of Ajuga iva (L.) and acarbose on the variation of postprandial glycaemia 887
in normal rats (A), with a representation in the form of areas under the curves (B). Values 888
are SEM means. (n=6). **** p < 0.001: compared to the control. 889
From these results, it is clear that the A. iva (L.) leaves could be used to treat post- 890
prandial hyperglycemia in diabetics. On the one hand, the aqueous extract is rich in nat- 891
ural bioactive molecules, (apeginen 7-O-glucoside, ferulic acid, coumaric acid, querce- 892
tin…) in addition to nutrients. On the other hand, the ease of their administration, the 893
availability, and the convenient cost of the plant make this plant a potential competitor to 894
the synthetic drug 'acarbose'. 895
4. Conclusion 896
In this work, chemical, biological and pharmacological study was carried out on the 897
leaves of A. iva (L.). The results obtained show a good correlation between the phenolic 898
compounds and the biological activities of the extracts studied. Thus, the chemical com- 899
position and the richness in minerals of our plant prove the important therapeutic effect 900
of A. iva (L.). The antidiabetic activity carried out on the decoction, the most commonly 901
used extract in traditional medicine, revealed a good inhibition of the activity of the en- 902
zyme α-amylase, thus decreasing postprandial hyperglycemia. These results could be ex- 903
plained by the presence of polyphenolic compounds in the decoction such as ferulic acid, 904
quercetin, coumaric acid and luteolin, etc. 905
Given these findings, the antidiabetic, antioxidant, and antimicrobial qualities iden- 906
tified during this study validate and support the Moroccan population's traditional usage 907
of A. iva (L.) leaves as a natural antidiabetic and/or antimicrobial remedy. 908
909
Authors’ contribution: All authors contributed to writing original draft, formal analysis, reviewing 910
and editing, data curation, funding acquisition, methodology, conceptualization and investigation 911
912
References 913
1. WHO Strategy for Traditional Medicine 2002-2005; Geneva, 2002; p. 65 914
2. Ladoh, Y.; Dibong, S.; Nyegue, M.; Djembissi, T.; Lenta, N.; Mpondo, M.; Yinyang, J.; Wansi, J. Antioxidant activity 915
of methanolic extracts of Phragmanthera capitata (Loranthaceae) harvested from Citrus sinensis. J. App. Bioscience. 916
2015, 84, 7636, doi:10.4314/jab.v84i1.9. 917
3. Loizzo, M.R.; Leporini, M.; Sicari, V.; Falco, T.; Pellicanò, T.M.; Tundis, R. Investigating the in Vitro 918
Hypoglycaemic and Antioxidant Properties of Citrus × Clementina Hort. Juice. Eur Food Res Technol 2018, 244, 919
523–534, doi:10.1007/s00217-017-2978-z. 920
4. Zbadi, R.; Mohti, H.; Moussaoui, F. Stress oxydatif : evaluation of the antioxidant power of some medicinal plants. 921
Medecine Therapeutique 2018, 24, 8. 922
5. Da Porto, C.; Porretto, E.; Decorti, D. Comparison of Ultrasound-Assisted Extraction with Conventional Extraction 923
Methods of Oil and Polyphenols from Grape (Vitis Vinifera L.) Seeds. Ultrasonics Sonochemistry 2013, 20, 1076– 924
1080, doi:10.1016/j.ultsonch.2012.12.002. 925
6. Alara, O.R.; Abdurahman, N.H.; Olalere, O.A. Ethanolic Extraction of Flavonoids, Phenolics and Antioxidants 926
from Vernonia Amygdalina Leaf Using Two-Level Factorial Design. Journal of King Saud University - Science 2020, 927
32, 7–16, doi:10.1016/j.jksus.2017.08.001. 928
7. Haleng, J.; Pincemail, J.; Defraigne, J.O.; Charlier, C.; Chapelle, J.P. Stress Oxydant, RMLg, 2007.Pdf. Rev Med Liege 929
2007, 62, 628–638. 930
8. B, D.; K, S.; B, L.; Rs, B.; Ps, B. A Modern Review of Diabetes Mellitus: An Annihilatory Metabolic Disorder. J In 931
Silico In Vitro Pharmacol 2017, 03, doi:10.21767/2469-6692.100014. 932

9. Sisodia, D.; Sisodia, D.S. Prediction of Diabetes Using Classification Algorithms. Procedia Computer Science 2018, 933
132, 1578–1585, doi:10.1016/j.procs.2018.05.122. 934
10. Reddy, P.; Duggar, B.; Butterworth, J. Blood Glucose Management in the Patient Undergoing Cardiac Surgery: A 935
Review. World J Cardiol 2014, 6, 1209–1217, doi:10.4330/wjc.v6.i11.1209. 936
11. Bouyahya, A.; El Omari, N.; Elmenyiy, N.; Guaouguaou, F.-E.; Balahbib, A.; Belmehdi, O.; Salhi, N.; Imtara, H.; 937
Mrabti, H.N.; El-Shazly, M.; et al. Moroccan Antidiabetic Medicinal Plants: Ethnobotanical Studies, Phytochemical 938
Bioactive Compounds, Preclinical Investigations, Toxicological Validations and Clinical Evidences; Challenges, 939
Guidance and Perspectives for Future Management of Diabetes Worldwide. Trends in Food Science & Technology 940
2021, 115, 147–254, doi:10.1016/j.tifs.2021.03.032. 941
12. Nash, R.J.; Kato, A.; Yu, C.-Y.; Fleet, G.W. Iminosugars as Therapeutic Agents: Recent Advances and Promising 942
Trends. Future Medicinal Chemistry 2011, 3, 1513–1521, doi:10.4155/fmc.11.117. 943
13. Tseng, P.; Ande, C.; Moremen, K.W.; Crich, D. Influence of Side Chain Conformation on the Activity of 944
Glycosidase Inhibitors. Angew Chem Int Ed 2023, 62, doi:10.1002/anie.202217809. 945
14. Brown, T.J.; Brainard, J.; Song, F.; Wang, X.; Abdelhamid, A.; Hooper, L. Omega -3, Omega-6, and Total Dietary 946
Polyunsaturated Fat for Prevention and Treatment of Type 2 Diabetes Mellitus: Systematic Review and Meta- 947
Analysis of Randomised Controlled Trials. BMJ 2019, l4697, doi:10.1136/bmj.l4697. 948
15. Chen, Y.-P.; Tsai, C.-W.; Shen, C.-Y.; Day, C.-H.; Yeh, Y.-L.; Chen, R.-J.; Ho, T.-J.; Padma, V.V.; Kuo, W.-W.; Huang, 949
C.-Y. Palmitic Acid Interferes with Energy Metabolism Balance by Adversely Switching the SIRT1 -CD36-Fatty 950
Acid Pathway to the PKC Zeta-GLUT4-Glucose Pathway in Cardiomyoblasts. The Journal of Nutritional 951
Biochemistry 2016, 31, 137–149, doi:10.1016/j.jnutbio.2016.01.007. 952
16. Israili, Z.H.; Lyoussi, B. Ethnopharmacology of the Plants of Genus Ajuga. Pak J Pharm Sci 2009, 22, 425–462. 953
17. Miara, M.D.; Hammou, M.A.; Aoul, S.H. Phytotherapy and taxonomy of spontaneous medicinal plants in the 954
region of Tiaret (Algeria). Phytothérapie 2013, 11, 206–218, doi:10.1007/s10298-013-0789-3. 955
18. Bendif, H.; Lazali, M.; Harir, M.; Miara, M.D.; Boudjeniba, M.; Venskutonis, P.R. Biological Screening of Ajuga Iva 956
Extracts Obtained by Supercritical Carbon Dioxide and Pressurized Liquid Extraction. J Med Bot 2017, 1, 33, 957
doi:10.25081/jmb.2017.v1.816. 958
19. Benkhnigue, O.; Ben Akka, F.; Salhi, S.; Fadli, M.; Douira, A.; Zidane, L. Catalog Of Medicinal Plants Used In The 959
Treatment Of Diabetes In The Al Haouz-Rhamna Region (Morocco). J Anim Plant Sci 2014, 23, 3539–3568. 960
20. Diafat, A.; Arrar, L.; Derradji, Y.; Bouaziz, F. Acute and Chronic Toxicity of the Methanolic Extract of Ajuga Iva in 961
Rodents. nternational Journal of Applied Research in Natural Products 2016, 9, 9–16. 962
21. El Amrani, F.; Rhallab, A.; Alaoui, T.; El Badaoui, K.; Chakir, S. Ethnopharmacological study of some plants used 963
in the treatment of diabetes in the Meknes-Tafilalet region (Morocco). Phytothérapie 2010, 8, 161–165, 964
doi:10.1007/s10298-010-0552-y. 965
22. El-Hilaly, J.; Lyoussi, B.; Wibo, M.; Morel, N. Vasorelaxant Effect of the Aqueous Extract of Ajuga Iva in Rat Aorta. 966
Journal of ethnopharmacology 2004, 93, 69–74. 967
23. El Rhaffari, L.; Zaid, A. Practice of phytotherapy in the south-east of Morocco (Tafilalet). Empirical knowledge for 968
a renewed pharmacopoeia. In Des sources du savoir aux médicaments du futur; Fleurentin, J., Pelt, J.-M., Mazars, G., 969
Eds.; IRD Éditions, 2002; pp. 293–318 ISBN 978-2-7099-1504-5. 970
24. Ghourri, M.; Zidane, L.; Douira, A. Use Of Medicinal Plants In The Treatment Of Diabetes In The Moroccan Sahara 971
(Tan-Tan). Journal of Animal and Plant Sciences 2013, 17, 2388–2411. 972
25. Jamila, F.; Mostafa, E. Ethnobotanical Survey of Medicinal Plants Used by People in Oriental Morocco to Manage 973
Various Ailments. Journal of ethnopharmacology 2014, 154, doi:10.1016/j.jep.2014.03.016. 974

26. Tahraoui, A.; El-Hilaly, J.; Israili, Z.H.; Lyoussi, B. Ethnopharmacological Survey of Plants Used in the Traditional 975
Treatment of Hypertension and Diabetes in South-Eastern Morocco (Errachidia Province). Journal of 976
Ethnopharmacology 2007, 110, 105–117, doi:10.1016/j.jep.2006.09.011. 977
27. Bouderbala, S.; Lamri-Senhadji, M.; Prost, J.; Lacaille-Dubois, M.A.; Bouchenak, M. Changes in Antioxidant 978
Defense Status in Hypercholesterolemic Rats Treated with Ajuga Iva. Phytomedicine 2008, 15, 453–461. 979
28. Boudjelal, A.; Siracusa, L.; Henchiri, C.; Sarri, M.; Abderrahim, B.; Baali, F.; Ruberto, G. Antidiabetic Effects of 980
Aqueous Infusions of Artemisia Herba-Alba and Ajuga Iva in Alloxan-Induced Diabetic Rats. Planta Medica 2015, 981
81, 696–704, doi:10.1055/s-0035-1546006. 982
29. Bouyahya, A.; El Omari, N.; Elmenyiy, N.; Guaouguaou, F.-E.; Balahbib, A.; El-Shazly, M.; Chamkhi, I. 983
Ethnomedicinal Use, Phytochemistry, Pharmacology, and Toxicology of Ajuga Iva (L.,) Schreb. Journal of 984
Ethnopharmacology 2020, 258, 112875, doi:10.1016/j.jep.2020.112875. 985
30. Chouitah, O.; Meddah, B.; Aoues, A.; Sonnet, P. Essential Oil from the Leaves of Ajuga Iva: Chemical Composition 986
and Antimicrobial Activity. Journal of Essential Oil Bearing Plants 2017, 20, 873–877. 987
31. Mouheb, S.; Khali, M.; Rouibi, A.; Saidi, F. Antimicrobial and Analgesic Activity of Aqueous Extract of Algerian 988
Ajuga Iva (l.) Schreb (Lamiaceae). Revue Agrobiologia 2018, 8, 863–870. 989
32. Bennaghmouch, L.; Hajjaji, N.; Gmira, N. Flavonoïdes d’Ajuga iva (L.) Schreb (Labiée). Actes Inst. Agron. Vet. 2002, 990
22, 25–30. 991
33. Medjeldi, S.; Bouslama, L.; Benabdallah, A.; Essid, R.; Haou, S.; Elkahoui, S. Biological Activities, and 992
Phytocompounds of Northwest Algeria Ajuga Iva (L) Extracts: Partial Identification of the Antibacterial Fraction. 993
Microbial Pathogenesis 2018, 121, 173–178, doi:10.1016/j.micpath.2018.05.022. 994
34. Coll, J.; Tandrón, Y.A. Neo-Clerodane Diterpenoids from Ajuga: Structural Elucidation and Biological Activity. 995
Phytochem Rev 2007, 7, 25–49, doi:10.1007/s11101-006-9023-3. 996
35. eFlore. Tela Botanica. 997
36. AFNOR « NF V03-402, Spices and herbs - Determination of water content - Training method ».; Afnor Editions, 1985; 998
37. AFNOR « NF V05-113, Fruits, vegetables and derived products - Mineralization of organic matter - Incineration method».; 999
Afnor Editions, 1972; 1000
38. Center of expertise in environmental analysis Analysis method: Determination of PH: Electrometric method; MA. 1001
100 – pH 1.1, Rev. 3; Ministry of Sustainable Development, Environment, Wildlife and Parks: Quebec , 2014; p. 11;. 1002
39. Bergeron, L. Effect of Soil Water Content on Lowbush Blueberry Fruit Yield and Qua lity /; University of Quebec at 1003
Chicoutimi: Chicoutimi, 1995; ISBN 978-1-4123-0614-0. 1004
40. Dey, P.; Harborne, J. Methods in Plant Biochemistry: Plant Phenolics. In; Academic Press Ltd.: London, 1989; Vol. 1005
1, p. 552 ISBN 978-0-12-461011-8. 1006
41. GIBBS DARNLEY, R. Chemotaxonomy of Flowering Plants: Four Volumes; McGill-Queen’s University Press: Montreal 1007
and London, 1974; Vol. 1;. 1008
42. Singleton, V.L.; Rossi, J.A. Colorimetry of Total Phenolics with Phosphomolybdic -Phosphotungstic Acid Reagents. 1009
Am J Enol Vitic. 1965, 16, 144–158. 1010
43. Chang, C.-C.; Yang, M.-H.; Wen, H.-M.; Chern, J.-C. Estimation of Total Flavonoid Content in Propolis by Two 1011
Complementary Colorimetric Methods. Journal of Food and Drug Analysis 2002, 10, 178–182, 1012
doi:https://doi.org/10.38212/2224-6614.2748. 1013
44. Sun, B.; Ricardo-da-Silva, J.M.; Spranger, I. Critical Factors of Vanillin Assay for Catechins and Proanthocyanidins. 1014
J. Agric. Food Chem. 1998, 46, 4267–4274, doi:10.1021/jf980366j. 1015

45. Wong, C.-C.; Li, H.-B.; Cheng, K.-W.; Chen, F. A Systematic Survey of Antioxidant Activity of 30 Chinese 1016
Medicinal Plants Using the Ferric Reducing Antioxidant Power Assay. Food Chemistry 2006, 97, 705–711, 1017
doi:10.1016/j.foodchem.2005.05.049. 1018
46. Oyaizu, M. Studies on products of browning reaction--antioxidative activities of products of browning reaction 1019
prepared from glucosamine. Eiyogaku zasshi = Japanese journal of nutrition 1986. 1020
47. Prieto, P.; Pineda, M.; Aguilar, M. Spectrophotometric Quantitation of Antioxidant Capacity through the 1021
Formation of a Phosphomolybdenum Complex: Specific Application to the Determination of Vitamin E. Analytical 1022
Biochemistry 1999, 269, 337–341, doi:10.1006/abio.1999.4019. 1023
48. Elshikh, M.; Ahmed, S.; Funston, S.; Dunlop, P.; McGaw, M.; Marchant, R.; Banat, I.M. Resazurin-Based 96-Well 1024
Plate Microdilution Method for the Determination of Minimum Inhibitory Concentration of Biosurfactants. 1025
Biotechnol Lett 2016, 38, 1015–1019, doi:10.1007/s10529-016-2079-2. 1026
49. Giweli, A.; Džamić, A.M.; Soković, M.; Ristić, M.S.; Marin, P.D. Antimicrobial and Antioxidant Activities of 1027
Essential Oils of Satureja Thymbra Growing Wild in Libya. Molecules 2012, 17, 4836–4850, 1028
doi:10.3390/molecules17054836. 1029
50. Reddy, Y.M.; Kumar, S.P.J.; Saritha, K.V.; Gopal, P.; Reddy, T.M.; Simal-Gandara, J. Phytochemical Profiling of 1030
Methanolic Fruit Extract of Gardenia Latifolia Ait. by LC-MS/MS Analysis and Evaluation of Its Antioxidant and 1031
Antimicrobial Activity. Plants 2021, 10, 545, doi:10.3390/plants10030545. 1032
51. Sarker, S.D.; Nahar, L.; Kumarasamy, Y. Microtitre Plate-Based Antibacterial Assay Incorporating Resazurin as 1033
an Indicator of Cell Growth, and Its Application in the in Vitro Antibacterial Screening of Phytochemicals. Methods 1034
2007, 42, 321–324, doi:10.1016/j.ymeth.2007.01.006. 1035
52. Prasannabalaji, N.; Muralitharan, G.; Sivanandan, R.; Kumaran, S.; Pugazhvendan, S. Antibacterial Activities of 1036
Some Indian Traditional Plant Extracts. Asian Pacific Journal of Tropical Disease 2012, 2, S291–S295, 1037
doi:10.1016/S2222-1808(12)60168-6. 1038
53. Tchoumtchoua, J.; Mouchili, O.R.; Ateba, S.B.; Zingue, S.; Halabalaki, M.; Mbanya, J.C.; Skaltsounis, A.-L.; Njamen, 1039
D. Safety Assessment of the Methanol Extract of the Stem Bark of Amphimas Pterocarpoides Harms: Acute and 1040
Subchronic Oral Toxicity Studies in Wistar Rats. Toxicology Reports 2014, 1, 877–884, 1041
doi:10.1016/j.toxrep.2014.10.003. 1042
54. Daoudi, N.E.; Bouhrim, M.; Ouassou, H.; Legssyer, A.; Mekhfi, H.; Ziyyat, A.; Aziz, M.; Bnouham, M. Inhibitory 1043
Effect of Roasted/ Unroasted Argania Spinosa Seeds Oil on α- Glucosidase, α-Amylase and Intestinal Glucose 1044
Absorption Activities. South African Journal of Botany 2020, 135, 413–420, doi:10.1016/j.sajb.2020.09.020. 1045
55. Boulfia, M.; Lamchouri, F.; Toufik, H. Mineral Analysis, In Vitro Evaluation of Alpha-Amylase, Alpha- 1046
Glucosidase, and Beta-Galactosidase Inhibition, and Antibacterial Activities of Juglans Regia L. Bark Extracts. 1047
Biomed Res Int 2021, 2021, 1585692, doi:10.1155/2021/1585692. 1048
56. Ribereau-Gayon P The Phenolic Compounds Of Plants.; Ed.Dunod.173-201.; 1968; 1049
57. Yokabd, D.; Mariamawit, Y.Y. Standardization of the Leaves of Ajuga Remota Benth. Ethiopian Pharmaceutical 1050
Journal 2022, Vol. 37 No. 1 (2021). 1051
58. Soro, T.Y.; Traore, F.; Sakande, J. Analgesic activity of the aqueous extract of Ximenia americana (Linnaeus) 1052
(Olacaceae). Comptes Rendus Biologies 2009, 332, 371–377, doi:10.1016/j.crvi.2008.08.022. 1053
59. Ammar, H.; Touihri, I.; Kholif, A.E.; M’Rabet, Y.; Jaouadi, R.; Chahine, M.; Marti, M.E. de H.; Vargas-Bello-Pérez, 1054
E.; Hosni, K. Chemical Composition, Antioxidant, and Antimicrobial Activities of Leaves of Ajuga Iva. Molecules 1055
2022, 27, 7102, doi:10.3390/molecules27207102. 1056

60. Ahmed, H.; Nawel, O. Phytochemical Studies And Antioxidant Activities Of Adiantum Capilus-Veneris, 1057
Lavandula Stoechas And Ajuga Iva. World Journal of Pharmaceutical Research 2016, 5, 25. 1058
61. Ammar, H.; Essid, S.; Brinsi, J.; Lopez, S. Chemical Composition and in Vitro Digestibility of Leaves of Tunisian 1059
Ajuga Iva. Options Méditerranéennes : Série A 2021, 5. 1060
62. Senhaji, S.; Lamchouri, F.; Boulfia, M.; Lachkar, N.; Bouabid, K.; Toufik, H. Mineral Composition, in Vitro 1061
Inhibitory Effects of α-Amylase, α-Glucosidase, β-Galactosidase Enzymes and Antibacterial Activity of Ajuga Iva 1062
Subsp. Pseudoiva (DC.) Bric. Biointerface Res Appl Chem 2022, 12, 2373–2391, doi:10.33263/BRIAC122.23732391. 1063
63. Ahmed, D.; Chaudhary, M.A. Medicinal and Nutritional Aspects of Various Trace Metals Determined In. J. App. 1064
Sci. Res 2009, 5, 864–869. 1065
64. Nema, N.K.; Maity, N.; Sarkar, B.K.; Mukherjee, P.K. Determination of Trace and Heavy Metals in Some 1066
Commonly Used Medicinal Herbs in Ayurveda. Toxicol Ind Health 2014, 30, 964–968, 1067
doi:10.1177/0748233712468015. 1068
65. Irawati, Y.; Kusnoputranto, H.; Achmadi, U.F.; Safrudin, A.; Sitorus, A.; Risandi, R.; Wangsamuda, S.; Setia Asih, 1069
P.B.; Syafruddin, D. Blood Lead Levels and Lead Toxicity in Children Aged 1 -5 Years of Cinangka Village, Bogor 1070
Regency. PLoS ONE 2022, 17, e0264209, doi:10.1371/journal.pone.0264209. 1071
66. Pourrut, B.; Shahid, M.; Dumat, C.; Winterton, P.; Pinelli, E. Lead Uptake, Toxicity, and Detoxification in Plants. 1072
In Reviews of Environmental Contamination and Toxicology Volume 213; Whitacre, D.M., Ed.; Reviews of 1073
Environmental Contamination and Toxicology; Springer New York: New York, NY, 2011; Vol. 213, pp. 113–136 1074
ISBN 978-1-4419-9859-0. 1075
67. FAO/OMS Evaluation of certain food additives and contaminants: Mercury, Lead and Cadmium; Geneva , 1972; p. 34;. 1076
68. Nuran Ercal, B.S.P.; Hande Gurer-Orhan, B.S.P.; Nukhet Aykin-Burns, B.S.P. Toxic Metals and Oxidative Stress 1077
Part I: Mechanisms Involved in Me-Tal Induced Oxidative Damage. CTMC 2001, 1, 529–539, 1078
doi:10.2174/1568026013394831. 1079
69. Kaur, S.; Kamli, M.R.; Ali, A. Role of Arsenic and Its Resistance in Nature. Can. J. Microbiol. 2011, 57, 769–774, 1080
doi:10.1139/w11-062. 1081
70. Shirkhanloo, H.; Mirzahosseini, S.A.H.; Shirkhanloo, N.; Moussavi-Najarkola, S.A.; Farahani, H. The Evaluation 1082
and Determination of Heavy Metals Pollution in Edible Vegetables, Water and Soil in the South of Tehran Province 1083
by GIS. Archives of Environmental Protection 2015, 41, 64–74, doi:10.1515/aep-2015-0020. 1084
71. Yoon, Y.; Lee, W.-M.; An, Y.-J. Phytotoxicity of Arsenic Compounds on Crop Plant Seedlings. Environ Sci Pollut 1085
Res 2015, 22, 11047–11056, doi:10.1007/s11356-015-4317-x. 1086
72. Organic Farming, Pest Control and Remediation of Soil Pollutants: Organic Farming, Pest Control and Remediation of Soil 1087
Pollutants; Lichtfouse, E., Ed.; Sustainable Agriculture Reviews; Springer Netherlands: Dordrecht, 2010; Vol. 1; 1088
ISBN 978-1-4020-9653-2. 1089
73. Orisakwe, O.E. 11 - Other Heavy Metals: Antimony, Cadmium, Chromium and Mercury. In Toxicity of Building 1090
Materials; Pacheco-Torgal, F., Jalali, S., Fucic, A., Eds.; Woodhead Publishing Series in Civil and Structural 1091
Engineering; Woodhead Publishing, 2012; pp. 297–333 ISBN 978-0-85709-122-2. 1092
74. Bolan, N.; Kumar, M.; Singh, E.; Kumar, A.; Singh, L.; Kumar, S.; Keerthanan, S.; Hoang, S.A.; El -Naggar, A.; 1093
Vithanage, M.; et al. Antimony Contamination and Its Risk Management in Complex Environmental Settings: A 1094
Review. Environment International 2022, 158, 106908, doi:10.1016/j.envint.2021.106908. 1095
75. Ben El Hadj Ali, I.; Bahri, R.; Chaouachi, M.; Boussaïd, M.; Harzallah-Skhiri, F. Phenolic Content, Antioxidant and 1096
Allelopathic Activities of Various Extracts of Thymus Numidicus Poir. Organs. Industrial Crops and Products 2014, 1097
62, 188–195, doi:10.1016/j.indcrop.2014.08.021. 1098

76. Kallel, F.; Driss, D.; Chaari, F.; Belghith, L.; Bouaziz, F.; Ghorbel, R.; Chaabouni, S.E. Garlic (Allium Sativum L.) 1099
Husk Waste as a Potential Source of Phenolic Compounds: Influence of Extracting Solvents on Its Antimicrobial 1100
and Antioxidant Properties. Industrial Crops and Products 2014, 62, 34–41, doi:10.1016/j.indcrop.2014.07.047. 1101
77. Khlifi, D.; Hamdi, M.; Hayouni, A.E.; Cazaux, S.; Souchard, J.P.; Couderc, F.; Bouajila, J. Global Chemical 1102
Composition and Antioxidant and Anti-Tuberculosis Activities of Various Extracts of Globularia Alypum L. 1103
(Globulariaceae) Leaves. Molecules 2011, 16, 10592–10603, doi:10.3390/molecules161210592. 1104
78. Fettach, S.; Mrabti, H.N.; Sayah, K.; Bouyahya, A.; Salhi, N.; Cherrah, Y.; El Abbes, F.M. Phenolic Content, Acute 1105
Toxicity of Ajuga Iva Extracts and Assessment of Their Antioxidant and Carbohydrate Digestive Enzyme 1106
Inhibitory Effects. South African Journal of Botany 2019, 125, 381–385, doi:10.1016/j.sajb.2019.08.010. 1107
79. Ouassou, H.; Bouhrim, M.; Bencheikh, N.; Addi, M.; Hano, C.; Mekhfi, H.; Ziyyat, A.; Legssyer, A.; Aziz, M.; 1108
Bnouham, M. In Vitro Antioxidant Properties, Glucose-Diffusion Effects, α-Amylase Inhibitory Activity, and 1109
Antidiabetogenic Effects of C. Europaea Extracts in Experimental Animals. Antioxidants 2021, 10, 1747, 1110
doi:10.3390/antiox10111747. 1111
80. Boutahiri, S.; Eto, B.; Bouhrim, M.; Mechchate, H.; Saleh, A.; Al kamaly, O.; Drioiche, A.; Remok, F.; Samaillie, J.; 1112
Neut, C.; et al. Lavandula Pedunculata (Mill.) Cav. Aqueous Extract Antibacterial Activity Improved by the 1113
Addition of Salvia Rosmarinus Spenn., Salvia Lavandulifolia Vahl and Origanum Compactum Benth. Life 2022, 1114
12, 328, doi:10.3390/life12030328. 1115
81. John, B.; Sulaiman, C.T.; George, S.; Reddy, V.R.K. Total Phenolics and Flavonoids in Selected Medicinal Plants 1116
from Kerala. Int. J. Pharm. Pharm. Sci 2014, 6, 406–408. 1117
82. Mathew, S.; Abraham, T.E. Ferulic Acid: An Antioxidant Found Naturally in Plant Cell Walls and Feruloyl 1118
Esterases Involved in Its Release and Their Applications. Critical Reviews in Biotechnology 2004, 24, 59–83, 1119
doi:10.1080/07388550490491467. 1120
83. Kikuzaki, H.; Hisamoto, M.; Hirose, K.; Akiyama, K.; Taniguchi, H. Antioxidant Properties of Ferulic Acid and Its 1121
Related Compounds. J. Agric. Food Chem. 2002, 50, 2161–2168, doi:10.1021/jf011348w. 1122
84. Raj, N.D.; Singh, D. A Critical Appraisal on Ferulic Acid: Biological Profile, Biopharmaceutical Challenges and 1123
Nano Formulations. Health Sciences Review 2022, 5, 100063, doi:10.1016/j.hsr.2022.100063. 1124
85. Narasimhan, A.; Chinnaiyan, M.; Karundevi, B. Ferulic Acid Exerts Its Antidiabetic Effect by Modulating Insulin- 1125
Signalling Molecules in the Liver of High-Fat Diet and Fructose-Induced Type-2 Diabetic Adult Male Rat. Appl. 1126
Physiol. Nutr. Metab. 2015, 40, 769–781, doi:10.1139/apnm-2015-0002. 1127
86. Zhang, Y.; Xie, Y.; Cheng, Z.; Xi, K.; Huang, X.; Kuang, F.; Wang, W.; Liu, T.; Guo, B.; Wu, S. Quercetin Ameliorates 1128
Memory Impairment by Inhibiting Abnormal Microglial Activation in a Mouse Model of Paradoxical Sleep 1129
Deprivation. Biochemical and Biophysical Research Communications 2022, 632, 10–16, doi:10.1016/j.bbrc.2022.09.088. 1130
87. Bule, M.; Abdurahman, A.; Nikfar, S.; Abdollahi, M.; Amini, M. Antidiabetic Effect of Quercetin: A Systematic 1131
Review and Meta-Analysis of Animal Studies. Food and Chemical Toxicology 2019, 125, 494–502, 1132
doi:10.1016/j.fct.2019.01.037. 1133
88. Lin, L.; Dong, Y.; Zhao, H.; Wen, L.; Yang, B.; Zhao, M. Comparative Evaluation of Rosmarinic Acid, Methyl 1134
Rosmarinate and Pedalitin Isolated from Rabdosia Serra (MAXIM.) HARA as Inhibitors of Tyrosinase and α - 1135
Glucosidase. Food Chemistry 2011, 129, 884–889, doi:10.1016/j.foodchem.2011.05.039. 1136
89. Septembre-Malaterre, A.; Boumendjel, A.; Seteyen, A.-L.S.; Boina, C.; Gasque, P.; Guiraud, P.; Sélambarom, J. 1137
Focus on the High Therapeutic Potentials of Quercetin and Its Derivatives. Phytomedicine Plus 2022, 2, 100220, 1138
doi:10.1016/j.phyplu.2022.100220. 1139
90. Calderon-Montano, J.M.; Burgos-Moron, E.; Perez-Guerrero, C.; Lopez-Lazaro, M. A Review on the Dietary 1140
Flavonoid Kaempferol. MRMC 2011, 11, 298–344, doi:10.2174/138955711795305335. 1141
91. Qian, S.; Gao, S.; Li, J.; Liu, S.; Diao, E.; Chang, W.; Liang, X.; Xie, P.; Jin, C. Effects of Combined Enzymatic 1142
Hydrolysis and Fed-Batch Operation on Efficient Improvement of Ferulic Acid and p-Coumaric Acid Production 1143
from Pretreated Corn Straws. Bioresource Technology 2022, 366, 128176, doi:10.1016/j.biortech.2022.128176. 1144
92. Li, J.; Zhao, N.; Xu, R.; Li, G.; Dong, H.; Wang, B.; Li, Z.; Fan, M.; Wei, X. Deciphering the Antibacterial Activity 1145
and Mechanism of P-Coumaric Acid against Alicyclobacillus Acidoterrestris and Its Application in Apple Juice. 1146
International Journal of Food Microbiology 2022, 378, 109822, doi:10.1016/j.ijfoodmicro.2022.109822. 1147
93. Amalan, V.; Vijayakumar, N.; Indumathi, D.; Ramakrishnan, A. Antidiabetic and Antihyperlipidemic Activity of 1148
P-Coumaric Acid in Diabetic Rats, Role of Pancreatic GLUT 2: In Vivo Approach. Biomedicine & Pharmacotherapy 1149
2016, 84, 230–236, doi:10.1016/j.biopha.2016.09.039. 1150
94. Toiu, A.; Mocan, A.; Vlase, L.; Pârvu, A.E.; Vodnar, D.C.; Gheldiu, A.-M.; Moldovan, C.; Oniga, I. Phytochemical 1151
Composition, Antioxidant, Antimicrobial and in Vivo Anti-Inflammatory Activity of Traditionally Used 1152
Romanian Ajuga Laxmannii (Murray) Benth. (“Nobleman’s Beard” – Barba Împăratului). Front. Pharmacol. 2018, 1153
9, 7, doi:10.3389/fphar.2018.00007. 1154
95. Boukada, F.; Meddah, B. Flavonoids from Aerial Part of Algerian Ajuga Iva (L.) Schreb.: The HPLC-UV Analysis 1155
and Antioxidant Capacity. Kragujevac J Science 2021, 23–34, doi:10.5937/KgJSci2143023B. 1156
96. Zahra, S.S.; Ahmed, M.; Qasim, M.; Gul, B.; Zia, M.; Mirza, B.; Haq, I. Polarity Based Characterization of 1157
Biologically Active Extracts of Ajuga Bracteosa Wall. Ex Benth. and RP-HPLC Analysis. BMC Complement Altern 1158
Med 2017, 17, 443, doi:10.1186/s12906-017-1951-5. 1159
97. Movahhedin, N.; Zengin, G.; Bahadori, M.B.; Sarikurkcu, C.; Bahadori, S.; Dinparast, L. Ajuga Chamaecistus 1160
Subsp. Scoparia (Boiss.) Rech.f.: A New Source of Phytochemicals for Antidiabetic, Skin -Care, and 1161
Neuroprotective Uses. Industrial Crops and Products 2016, 94, 89–96, doi:10.1016/j.indcrop.2016.08.028. 1162
98. Pang, Y.; Ahmed, S.; Xu, Y.; Beta, T.; Zhu, Z.; Shao, Y.; Bao, J. Bound Phenolic Compounds and Antioxidant 1163
Properties of Whole Grain and Bran of White, Red and Black Rice. Food Chemistry 2018, 240, 212–221, 1164
doi:10.1016/j.foodchem.2017.07.095. 1165
99. Yuwang, P.; Sulaeva, I.; Hell, J.; Henniges, U.; Böhmdorfer, S.; Rosenau, T.; Chitsomboon, B.; Tongta, S. Phenolic 1166
Compounds and Antioxidant Properties of Arabinoxylan Hydrolysates from Defatt ed Rice Bran: Phenolic and 1167
Antioxidant Properties of Rice Bran Arabinoxylan Hydrolyzates. J. Sci. Food Agric 2018, 98, 140–146, 1168
doi:10.1002/jsfa.8448. 1169
100. Makni, M.; Haddar, A.; Kriaa, W.; Zeghal, N. Antioxidant, Free Radical Scavenging, and Antimicrobia l Activities 1170
of Ajuga Iva Leaf Extracts. International Journal of Food Properties 2013, 16, 756–765, 1171
doi:10.1080/10942912.2011.561465. 1172
101. Ksouri, R.; Megdiche, W.; Falleh, H.; Trabelsi, N.; Boulaaba, M.; Smaoui, A.; Abdelly, C. Influence of Biological, 1173
Environmental and Technical Factors on Phenolic Content and Antioxidant Activities of Tunisian Halophytes. 1174
Comptes Rendus Biologies 2008, 331, 865–873, doi:10.1016/j.crvi.2008.07.024. 1175
102. Djeridane, A.; Yousfi, M.; Nadjemi, B.; Boutassouna, D.; Stocker, P.; Vidal, N. Antioxidant Activity of Some 1176
Algerian Medicinal Plants Extracts Containing Phenolic Compounds. Food Chemistry 2006, 97, 654–660, 1177
doi:10.1016/j.foodchem.2005.04.028. 1178
103. Cai, Y.; Luo, Q.; Sun, M.; Corke, H. Antioxidant Activity and Phenolic Compounds of 112 Traditional Chinese 1179
Medicinal Plants Associated with Anticancer. Life Sciences 2004, 74, 2157–2184, doi:10.1016/j.lfs.2003.09.047. 1180
104. Mouheb, S.; Khali, M.; Rouibi, A.; Saidi, F. Antimicrobial and Analgesic Activity of Aqueous Extract of Algerian 1181
Ajuga Iva (L.) Schreb (Lamiaceae). AgroBiologia 2018, 8, 863–870. 1182
105. Zerroug, M.M.; Zouaghi, M.; Boumerfeg, S.; Baghiani, A.; Nicklin, J.; Arrar, L. Antibacterial Activity Of Extracts 1183
Of Ajuga Iva, And Teucrium Polium. Adv. Environ. Biol. 2011, 5, 491–495. 1184
106. Ayari, B.; Riahi, L.; Ziadi, S.; Chograni, H.; Mliki, A. Evaluation Of Antioxidant And Antimicrobial Activities Of 1185
Tunisian Ajuga Iva L. Essential Oils. Revue de la Faculté des Sciences de Bizerte 2013, 11, 203–210. 1186
107. Chanwitheesuk, A.; Teerawutgulrag, A.; Kilburn, J.D.; Rakariyatham, N. Antimicrobial Gallic Acid from 1187
Caesalpinia Mimosoides Lamk. Food Chemistry 2007, 100, 1044–1048, doi:10.1016/j.foodchem.2005.11.008. 1188
108. Penna, C.; Marino, S.; Vivot, E.; Cruañes, M.C.; de D. Muñoz, J.; Cruañes, J.; Ferraro, G.; Gutkind, G.; Martino, V. 1189
Antimicrobial Activity of Argentine Plants Used in the Treatment of Infectious Diseases. Isolation of Active 1190
Compounds from Sebastiania Brasiliensis. Journal of Ethnopharmacology 2001, 77, 37–40, doi:10.1016/S0378- 1191
8741(01)00266-5. 1192
109. Cowan, M.M. Plant Products as Antimicrobial Agents. CLIN. MICROBIOL. REV. 1999, 12, 564–582, 1193
doi:10.1128/CMR.12.4.564. 1194
110. Xu, J.; Zhou, F.; Ji, B.-P.; Pei, R.-S.; Xu, N. The Antibacterial Mechanism of Carvacrol and Thymol against 1195
Escherichia Coli. Letters in Applied Microbiology 2008, 47, 174–179, doi:10.1111/j.1472-765X.2008.02407.x. 1196
111. El-Hilaly, J.; Tahraoui, A.; Israili, Z.H.; Lyoussi, B. Acute Hypoglycemic, Hypocholesterolemic And 1197
Hypotriglyceridemic Effects Of Continuous Intravenous Infusion Of A Lyophilised Aqueous Extract Of Ajuga Iva 1198
L. Schreber Whole Plant In Streptozotocin-Induced Diabetic Rats. Pak. J. Pharm. Sci. 2007, 20, 261–268. 1199
112. Bennaghmouch, L.; Hajjaji, N.; Zellou, A.; Cherrah, Y. [Pharmacological study of Ajuga iva]. Ann Pharm Fr 2001, 1200
59, 284. 1201
113. Bouyahya, A.; El Omari, N.; Elmenyiy, N.; Guaouguaou, F.-E.; Balahbib, A.; El-Shazly, M.; Chamkhi, I. 1202
Ethnomedicinal Use, Phytochemistry, Pharmacology, and Toxicology of Ajuga Iva (L.,) Schreb. Journal of 1203
Ethnopharmacology 2020, 258, 112875, doi:10.1016/j.jep.2020.112875. 1204
114. Rouibi, A.; Khali, M.; Saidi, F. Antispasmodic and Hypoglycemic Effects of Aqueous Extract of Ajuga Iva L. in 1205
Induced Diabetic Rats. Bulletin UASVM Animal Science and Biotechnologies 2013, 70, 339–343. 1206
115. Taleb-Senouci, D.; Lacaille-Dubois, M.A.; Bouchenak, M. Ajuga Iva Aqueous Extract Improves Reverse Cholesterol 1207
Transport in Streptozotocin-Induced Diabetic Rat. Journal of Pharmacy and Pharmacology 2012, 64, 1188–1194, 1208
doi:10.1111/j.2042-7158.2012.01501.x. 1209
116. Boudjelal, A.; Siracusa, L.; Henchiri, C.; Sarri, M.; Abderrahim, B.; Baali, F.; Ruberto, G. Antidiabetic Effects of 1210
Aqueous Infusions of Artemisia Herba-Alba and Ajuga Iva in Alloxan-Induced Diabetic Rats. Planta Med 2015, 81, 1211
696–704, doi:10.1055/s-0035-1546006. 1212
117. Cazarolli, L.H.; Kappel, V.D.; Pereira, D.F.; Moresco, H.H.; Brighente, I.M.C.; Pizzolatti, M.G.; Silva, F.R.M.B. Anti- 1213
Hyperglycemic Action of Apigenin-6-C-β-Fucopyranoside from Averrhoa Carambola. Fitoterapia 2012, 83, 1176– 1214
1183, doi:10.1016/j.fitote.2012.07.003. 1215
118. Cazarolli, L.H.; Folador, P.; Moresco, H.H.; Brighente, I.M.C.; Pizzolatti, M.G.; Silva, F.R.M.B. Stimulatory Effect 1216
of Apigenin-6-C-β-l-Fucopyranoside on Insulin Secretion and Glycogen Synthesis. European Journal of Medicinal 1217
Chemistry 2009, 44, 4668–4673, doi:10.1016/j.ejmech.2009.07.001. 1218
119. Cazarolli, L.H.; Folador, P.; Moresco, H.H.; Brighente, I.M.C.; Pizzolatti, M.G.; Silva, F.R.M.B. Mechanism of 1219
Action of the Stimulatory Effect of Apigenin-6-C-(2″-O-α-l-Rhamnopyranosyl)-β-l-Fucopyranoside on 14C- 1220
Glucose Uptake. Chemico-Biological Interactions 2009, 179, 407–412, doi:10.1016/j.cbi.2008.11.012. 1221

120. Uchiyama, M.; Yoshida, T. Effect of Ecdysterone on Carbohydrate and Lipid Metabolism. In Invertebrate 1222
Endocrinology and Hormonal Heterophylly; Burdette, W.J., Ed.; Springer Berlin Heidelberg: Berlin, Heidelberg, 1974; 1223
pp. 401–416 ISBN 978-3-642-65771-9. 1224
121. Chen, Q.; Xia, Y.; Qiu, Z. Effect of Ecdysterone on Glucose Metabolism in Vitro. Life Sciences 2006, 78, 1108–1113, 1225
doi:10.1016/j.lfs.2005.06.031. 1226
122. Lebovitz, H.E. Alpha-Glucosidase Inhibitors. Endocrinology and Metabolism Clinics of North America 1997, 26, 539– 1227
551, doi:10.1016/s0889-8529(05)70266-8. 1228
123. Kee, K.T.; Koh, M.; Oong, L.X.; Ng, K. Screening Culinary Herbs for Antioxidant and α-Glucosidase Inhibitory 1229
Activities. International Journal of Food Science & Technology 2013, 48, 1884–1891, doi:10.1111/ijfs.12166. 1230
124. Evert, A.B.; Boucher, J.L.; Cypress, M.; Dunbar, S.A.; Franz, M.J.; Mayer-Davis, E.J.; Neumiller, J.J.; Nwankwo, R.; 1231
Verdi, C.L.; Urbanski, P.; et al. Nutrition Therapy Recommendations for the Management of Adults with Diabetes. 1232
Diabetes Care 2014, 37 Suppl 1, S120-143, doi:10.2337/dc14-S120. 1233
125. Bouhrim, M.; Ouassou, H.; Boutahiri, S.; Daoudi, N.E.; Mechchate, H.; Gressier, B.; Eto, B.; Imtara, H.; A. Alotaibi, 1234
A.; Al-zharani, M.; et al. Opuntia Dillenii (Ker Gawl.) Haw., Seeds Oil Antidiabetic Potential Using In Vivo, In 1235
Vitro, In Situ, and Ex Vivo Approaches to Reveal Its Underlying Mechanism of Action. Molecules 2021, 26, 1677, 1236
doi:10.3390/molecules26061677. 1237
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Article 1

Phytochemical profile, antioxidant, antimicrobial and antidia- 2

betic activities of Ajuga iva (L.) 3

Life 2023, 13, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/life

2. Materials and methods 108

2.1. Chemicals products 109

2.2. Plant material 113

Table 1. : Ajuga's taxonomic position in the family of plants [35] . 121

2.3. Microbial material 123

Table 2. List of bacterial and fungal strains tested. 127

Bacterial strains Reference Fungal strains Reference

Escherichia coli BLSE 2DT2057 Candida tropicalis Ct

2.4. Animals 130

2.5. Preparation of extracts 136

2.5.1. Preparation of the decocted extract 137

2.5.2. Preparation of aqueous and hydroethanolic extracts 143

2.6. Parameters evaluated of plant material 149

2.6.1. Water content 150

2.6.2. Ashes 160

2.6.3. pH determination 173

2.6.4. Titratable acidity (TA) 177

2.8. Phytochemical study of Ajuga iva (L) leaves extracts. 220

2.8.2. Dosage of phenolic compounds 235

2.8.2.1. Determination of Total Polyphenol Content (TPC) 236

D : Dilution factor 255

2.8.2.2. Determination of Flavonoid Content (FC) 258

mextract : masse of extract 271

2.8.2.3. Determination of Condensed Tannin Content (CT) 272

2.9. Evaluation of antioxidant power 287

2.9.1. Radical scavenging activity (DPPH*) 290

2.9.2. Ferric Reducing Antioxidant Power (FRAP) 305

2.9.3. Total antioxidant activity (CAT) 318

2.10. Antimicrobial activity 328

2.11. Acute toxicity of decoction of Ajuga iva (L.) 346

2.12. Antihyperglycemic effect of decoction of Ajuga Iva (L.) 358

2.13.1. In-vitro test 370

2.13.2. In-vivo test 391

2.14. Statistical analysis 401

3. Results and discussion 407

3.1. Quality control of Ajuga iva (L.) 408

3.1.1. Water content 412

3.1.2. pH determination 424

3.1.3. Titratable acidity 430

3.1.4. Ash content 435

Plant Moisture content (%) pH Ash (%) Titratable acidity (%)

3.2. Quantification of metals 449

Ajuga iva (L.) As Cr Sb Pb Cd Fe Cu Ti

3.3. Phytochemical screening 517

Table 5. Phytochemical screening of Ajuga iva (L.) leaves. 528

Chemical groups Observations

Polyphenol Flavonoid Catechic tannin

Table 7. Chemical composition of Ajuga iva (L.) decoction by LC/UV/MS. 609

3.6. Antioxidant activity of different Ajuga iva (L.) extracts 611

DPPH* CAT FRAP

TC 655 -0,81 -0,94 1,0 CAT 0,2

3.8. Antimicrobial Activity of Ajuga iva Extracts 688

3.8.1. Antifungal activity 689

MIC Extracts (mg/mL)

3.8.2. Antibacterial activity 717

Bacteria Extracts (mg/mL)

Decocted Aqueous extract Hydroethanolic

3.10. Antihyperglycemic effect of decocted extract of Ajuga iva (L.) 772

Figure 4. variation in 820