Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : August 22, 2022
Accepted : September 14, 2022
Published online : September 30, 2022
doi:10.24659/gsr.9.3_175

Original article

Antiglycative effect of extract and phenolic compounds fromholy basil (Ocimum tenuiflorum)
Kento Kunihiro 1, 2), Tai Kaneshima 3), Takao Myoda 3)
1) Graduate School of Bioindustry, Tokyo University of Agriculture, Hokkaido, Japan
2) Albion Co. Ltd., Tokyo, Japan
3) Department of Food, Aroma and Cosmetic Chemistry, Tokyo University of Agriculture, Hokkaido, Japan

Abstract

Aim: Glycation reactions involve the binding of sugars to amino groups of proteins to form advanced glycation end products(AGEs) via glycation intermediates. AGEs induce inflammatory and protein dysfunctions, leading to diabetes and skin aging.
The aim of this study was to evaluate the anti-glycation effect of holy basil (Ocimum tenuiflorum) leaf extract and identify thecompounds responsible for these effects.
Methods: The crude extract was obtained by hot water extraction of dried holy basil leaves. The total polyphenol andflavonoid contents and antioxidant activities of the crude extract were determined. Phenolic compounds were isolated fromthe crude extract using various chromatographic methods. The effect of the crude extract and isolated compounds on theformation of fluorescent AGEs, N ε-(carboxymethyl) lysine (CML), 3-deoxyglucosone (3-DG), glyoxal (GO), and methylglyoxal(MGO) were examined.
Results: The crude extract of holy basil was rich in polyphenols, most of which were flavonoids. Luteolin 5-O-glucoside(1), luteolin 7-O-glucuronide (2), (−)-rabdosiin (3), apigenin 7-O-glucuronide (4), rosmarinic acid (5), globoidnan A (6),and salvianolic acid A (7) isolated from the crude extract inhibited the formation of fluorescent AGEs, CML, 3-DG, GO,
and MGO. In particular, compounds 3, 5, 6, and 7 were potent antiglycation compounds. The crude extract also showed highantioxidant activities.
Conclusions: Phenolic compounds of holy basil were found to be the active components endowing antiglycative effects.
One of the antiglycation mechanisms of these polyphenols could be related to the antioxidant action of holy basil.

KEY WORDS: antiglycative effect, advanced glycation end products (AGEs),		

holy basil (Ocimum tenuiflorum), polyphenols

Introduction
Glycation is a non-enzymatic reaction between reducing
sugars and amino groups in proteins, lipids, and nucleic acids
and produces advanced glycation end products (AGEs) 1).
Accumulation of AGEs, such as N ε-(carboxymethyl) lysine(CML), in dermal tissue influences glycation stress in theskin. AGEs partially accumulate in the skin by binding tocollagen proteins. Lysine and arginine, the amino acids
in collagen, can be glycated to AGEs to form crosslinks
between glycated collagen. Glycation of collagen inducesskin aging, leading to the loss of skin elasticity and wrinkle
formation 2). Glycation is an aging process related to oxidative
stress and is also associated with diabetic complications,atherosclerosis, neuropathies, and Alzheimer's disease 3, 4).
Furthermore, diseases caused by the glycation reaction invivo result from oxidative and inflammatory stresses due
to reactive oxygen species, which induce structural andfunctional protein alterations, cellular dysfunction, apoptosis,
and organ injuries 5). Thus, glycation is associated withvarious diseases and aging. Therefore, inhibition of glycation
is important for protection against aging and for maintaining
homeostasis.
The genus Ocimum, belonging to the family Labiatae,
consists of 30 species that are distributed in tropical andsubtropical regions. Holy basil (Ocimum tenuiflorum)has traditionally been used in India and Sri Lanka for the

Correspondence author: Tai Kaneshima, PhD
Department of Food, Aroma and Cosmetic Chemistry, Tokyo University of Agriculture
196 Yasaka, Abashiri-shi, Hokkaido 099-2493, Japan
TEL: +81-152-48-3871 FAX: +81-152-48-2531 e-mail: tk203897@nodai.ac.jp
Co-authors: Kunihiro K, k_kunihiro@albion.co.jp; Myoda T, t1myouda@nodai.ac.jp_ 175 _

Glycative Stress Research 2022; 9 (3) : 175-185(c) Society for Glycative Stress ResearchAntiglycative-active Substances of Holy Basiltreatment of various diseases. This plant is used to prepare
herbal tea or dried powder and treat colds, headaches,coughs, heart disease, and skin diseases 6). A previous study
suggested that the essential oils of holy basil leaves havepotent antioxidant activity 7). Holy basil also contains many
non-volatile compounds, including saponins, flavonoids,
and tannins 8). Zhao et al. showed that polyphenols, i.e.
protocyanidins, condensed tannins, and flavonoids, play animportant role in antiglycation activity 9). Holy basil can be
expected to have a high inhibitory effect on glycation because
phenolic compounds such as luteolin, apigenin, orientin,
eugenol, caffeic acid, and rosmarinic acid have been reported
as the main bioactive constituents 10, 11). The aim of this study
was to evaluate the antiglycative effect of the extract fromholy basil leaves, quantify the phenolic compounds andtheir antioxidant activities, and investigate the formation offluorescent AGEs, CML, and glycation intermediates.

Material and methods

1) Chemicals and reagents

Gallic acid, methanol (MeOH), Folin-Ciocalteu reagent,sodium hydroxide (NaOH), (+)-catechin, sodium nitrite,
aluminum trichloride, acetic acid, sodium acetate trihydrate,methanol- d 4 (MeOD), potassium persulfate, acetonitrile,
formic acid, rosmarinic acid, salvianolic acid A, glucose,
sodium hydrogen phosphate, sodium dihydrogen phosphate,aminoguanidine, 2, 3-butanedione, perchloric acid, sodiumhydrogen carbonate, 2, 3-diaminonaphthalene, ethyl acetate,glyoxal (GO), methylglyoxal (MGO) and ethanol (EtOH)
were purchased from Fujifilm Wako Pure Chemicals (Osaka,
Japan). 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2, 2’-azinobis (3-ethylbenzothiazoline-6 -sulfonic acid) (ABTS), andbovine serum albumin (BSA) were obtained from Sigma
Aldrich (St. Louis, USA). 3-deoxyglucosone (3-DG) was
purchased from Dojindo Laboratory (Kumamoto, Japan).
Luteolin 5-O-glucoside, luteolin 7-O-glucronide, and apigenin
7-O-glucronide were purchased from Namiki Shoji (Tokyo,
Japan). (−)-Rabdosiin was obtained from MedChemExpress(NJ, USA).

2) General experimental methods
The UV (ultra violet) spectra were acquired on a V-570
UV/Vis spectrometer (Jasco, Tokyo). 1 H and 13 C nuclear
magnetic resonance (NMR) spectra were recorded on a
ECZ-600 spectrometer (JEOL, Tokyo) at 600 and 125 MHz,
respectively. Chemical shifts are expressed in δ (ppm) andreferenced to the residual solvent peaks. The optical rotations
were measured using a Jasco P-1030 instrument. Highperformance liquid chromatography (HPLC) was performed
on a Jasco LC-2000 Plus HPLC system equipped with an
MD-2010 Plus photodiode array (PDA) detector and an
Atlantis T3 column (3 μm, 150 mm × 4.6 mm i.d., Waters,
Milford, MA, USA) using an acetonitrile-H2O-formic acidsolvent system. Mass spectra were obtained using liquid
chromatography time-of-flight mass spectrometry (LCTOF-MS). The LC-TOF-MS was performed on an Agilent
1200 Infinity HPLC and Agilent 6540 Agilent Ultra-HighDefinition Accurate-Mass Q-TOF LC/MS instrument. The

mass spectra were recorded in the negative electrosprayionization (ESI) mode. Preparative HPLC was performed
using a LC-8A pump (Shimadzu, Kyoto, Japan), UV-4570
detector (Jasco), and Inertsil ODS-4 column (3 μm, 250 mm
× 20 mm i.d., GL Sciences, Tokyo). The samples were eluted
at a flow rate of 6.0 mL/min using 20 % ~ 27 % acetonitrile
containing 0.1% acetic acid as a mobile phase. Signals wererecorded at 280 nm.

3) Plant materials and extraction
Holy basil (Ocimum tenuiflorum) fresh leaves werecollected in a medicinal garden at the Institute of Traditional
Plants (Negombo, Sri Lanka). The seedlings were purchasedfrom the Department of Agriculture (Peradeniya, Sri Lanka)
under the Ministry of Agriculture, which identified by theIndustrial Technology Institute (Colombo, Sri Lanka). Then,the plant was cultivated for at least six months. The collected
leaves were washed, and dried at 25 °C and 50 % ~ 70 %humidity for two weeks. The dried leaves of holy basil (50 g)
were extracted with distilled water (500 mL) at 100 °C for 1 h.
Then, the extract was filtered, evaporated, and lyophilized toobtain the crude extract (11.6 g).

4) Determination of total polyphenol content
The total polyphenol content was determined using the
Folin-Ciocalte method 12). The crude extract was adjusted to
0.5 mg/mL with 50 % MeOH and used as the sample solution.
The sample solution (20 μL) was mixed with 1 N FolinCiocalteu reagent (40 μL). Then, 1 N NaOH (100 μL) was
added to the mixture and incubated for 15 min, followedby centrifugation at 20,000 × g and 4 °C for 5 min. The
supernatant was dispensed into 96 well plate, and theabsorbance was measured at 734 nm using a microplate
reader (SpectraMax i3x, Molecular Devices, CA, USA). The
total polyphenol content was expressed as mg gallic acidequivalents/g of the sample. The assays were performed intriplicate.

5) Determination of total flavonoid content
The total flavonoid content was determined using thealuminum chloride colorimetric assay 13). The crude extract
was adjusted to 0.5 mg/mL with 50 % MeOH and used as the
sample solution. The sample solution (125 μL) was mixed
with 5 % sodium nitrite (40 μL) and distilled water (625 μL)
and incubated for 5 min. Next, 10 % aluminum trichloride(250 μL) was added and incubated for 6 min, followed by theaddition of 1 N NaOH (250 μL) and distilled water (140 μL).
The mixture was vortexed and the absorbance at 510 nm
was measured using a microplate reader. The total flavonoid
content was expressed as mg (+)-catechin equivalents pergram of the sample. The assays were performed in triplicate.

6) 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical
scavenging assay

The DPPH radical scavenging activity was measured asreported previously 7). The crude extract was adjusted to 1 ~100 μg/mL with 50 % MeOH and used as the sample solution.
The sample solution (40 μL) was mixed with 100 mM acetate_ 176 _Glycative Stress Researchbuffer (40 μL, pH 5.5), and then a 0.5 mM DPPH• solution
in EtOH (20 μL) was added. The reaction mixture wasvortexed and incubated in the dark for 30 min at 30 °C, after
which the absorbance was measured at 517 nm (A 517) using a
microplate reader. The DPPH radical scavenging activity wascalculated using the following equation:
DPPH radical scavenging activity (%)= [(A 517 (control) − A 517 (sample)) / A 517 (control)] × 100
IC 50 (50 % inhibitory concentration) values were
calculated from the percentage decrease relative to the sample
concentration. The assays were performed in triplicate. Gallic
acid and ascorbic acid were used as positive controls.

7) 2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonicacid) (ABTS) radical cation scavenging assay

The ABTS radical cation scavenging activity wasmeasured as reported previously 7). First, 10 mL of 7 mM
ABTS• solution was mixed with 176 μL of 140 mM potassium
persulfate, and the mixture was allowed to stand in the dark
at 25 °C for 12 ~ 16 h. The solution was then diluted 30-foldwith EtOH, and the absorbance of this ABTS• solution at
734 nm was measured to be approximately 0.700 using aspectrophotometer. The crude extract was adjusted to 1 ~ 200
μg/mL with 50% MeOH and used as the sample solution.
The sample solution (20 μL) was then added to the ABTS•
solution (200 μL). The mixture was vortexed and incubatedfor 4 min at 37 °C, following which the absorbance wasread at 734 nm (A 734) using a microplate reader. The ABTS
radical cation scavenging activity was calculated using thefollowing equation:
ABTS radical cation scavenging activity (%)= [(A 734 (control) − A 734 (sample)) / A 734 (control)] × 100
IC 50 values were calculated from the percentage
decrease relative to the sample concentration. The assays
were performed in triplicate. Gallic acid and ascorbic acid
were used as positive controls.

8) Superoxide dismutase (SOD)-like activity
The SOD-like activity was evaluated using the SOD
Assay Kit-WST (Dojindo Laboratory, Kumamoto, Japan)
according to the protocol. The crude extract was adjusted
to 1 ~ 50 μg/mL with 50% MeOH and used as the sample
solution. The sample solution (20 μL) and 200 μL of WST
working solution were mixed with enzyme working solution(20 μL). The mixture was vortexed and incubated for 20 min
at 37 °C, following which the absorbance was read at 450 nm(A 450) using a microplate reader. The antioxidant activity
was expressed as the percentage inhibition, as follows:
Inhibition (%)= [(A 450 (control) − A 450 (sample)) / A 450 (control)] × 100
IC 50 values were calculated from the percentage
decrease relative to the sample concentration. The assays
were performed in triplicate. Gallic acid and ascorbic acid
were used as positive controls.

9) Isolation of phenolic compounds
The crude extract (8.0 g) was dissolved in distilledwater and applied to a DIAION HP-20 column (Mitsubishi
Chemical, Tokyo; 35 g; 330 mm × 30 mm i.d.) equilibrated
with distilled water. The column was eluted with a H2OMeOH solvent system (300 mL) and three fractions, namely,
H2O Fr. (5.40 g), 50 % MeOH Fr. (1.84 g), and 100 % MeOH
Fr. (0.50 g), were obtained. The 50 % MeOH Fr. sample(1.0 g) was further separated on a Sephadex LH-20 column(Cytiva Sweden AB, Upsala, Sweden; 25 g; 300 mm × 30 mm
i.d.) using H 2O/MeOH mixture (100 :0, 75:25, 50:50, 25:75,
and 0:100 v/v; 200 mL). The fractions collected using 25 %~ 100 % MeOH were dissolved in 20 % ~ 27% acetonitrile,and the soluble portion was subjected to preparative HPLC.
Seven phenolic compounds were isolated from each of thefractions. Among the seven compounds, compound 1 (6.1mg) was isolated from 75% MeOH Fr., compound 2 (66.3mg), 3 (7.1 mg), 4 (6.8 mg), and 5 (23.8 mg) were isolated
from 25% MeOH Fr., and compound 6 (6.2 mg) and 7 (3.5mg) were isolated from the 100 % MeOH fraction. The
purities of the isolated compounds were confirmed by HPLC.
The structures of compounds 1 ~ 7 (Fig. 1 ) obtained fromthe crude extract were analyzed by LC-TOF-MS, 1 H NMR
spectroscopy, 13 C NMR spectroscopy, UV spectroscopy,
and specific optical rotation measurement. Compounds
were identified by a direct comparison of the HPLC
chromatograms with those of the standards.

10) Preparation of glycated proteins
The glycated proteins were prepared in accordance witha previously reported method 14). The BSA-glucose glycation
model was used to investigate the effect of the crude extractof holy basil and the compounds isolated from it uponglycation. BSA was added to a final concentration of 10
mg/mL to a 100 mg/mL glucose solution prepared in 0.1 M
phosphate-buffered saline (PBS, pH 7.2), and the mixture
was incubated at 60 °C for seven days (solution A). As abackground, a 10 mg/mL BSA solution without glucose wasprepared (solution B). To clarify the effects of the crude
extract and isolated compounds, samples of the indicatedconcentrations were added to the reaction mixture in
the presence or absence of glucose (solutions C and D,
respectively). Aminoguanidine was used as a positive control.
Aliquots of the reaction mixtures were then assayed for theformation of AGE and AGE intermediates. The reaction
mixtures were kept at −20 °C until analysis.

11) Measurement of AGE-derived fluorescence
AGE formation was measured using a fluorescencemicroplate reader at excitation and emission wavelengths of
370 and 440 nm, respectively 15). The inhibitory activity against
fluorescent AGE formation of the crude extract, isolated
compound, and aminoguanidine was calculated as following
equation and the IC 50 values also were obtained from the plotof the inhibitory activity relative to the sample concentration.
Inhibitory activity of fluorescent AGE formation (%)= {[fluorescence of (solution A − solution B) − (solution C −solution D)] / fluorescence of (solution A − solution B)} × 100_ 17 7 _Antiglycative-active Substances of Holy Basil

Luteolin 5-O-glucoside (1)Luteolin 7-O-glucronide (2)(−)-Rabdosiin (3)Apigenin 7-O-glucronide (4)Rosmarinic acid (5)Globoidnan A (6)Fig. 1. Chemical structures of the phenolic compounds isolated from holy basil.
Fig. 1. Chemical structures of the phenolic compounds isolated from holy basil.
Salvianolic acid A (7)Fig. 1. Chemical structures of the phenolic compounds isolated from holy basil.
Fig. 1. Chemical structures of the phenolic compounds isolated from holy basil.
Fig. 1. Chemical structures of the phenolic compounds isolated from holy basil.

_ 178 _Glycative Stress Research

Results12) Measurement of CML
CML is a non-fluorescent AGE and the most abundant
product of the glycation reaction. CML produced in thereaction mixtures was quantified by ELISA (enzymelinked immuno sorbent assay) using the CircuLex CML/N ε(carboxymethyl) lysine ELISA Kit (Medical and Biological
Laboratories, Tokyo, Japan), following the manufacturer’s
instructions. To prepare the test solution, the reaction mixture
was diluted 250-fold with 0.1 M PBS. The CML content
showed as the percentage of CML formation compared tothat of controls without sample addition.

13) Measurement of AGE intermediates
3-DG, GO, and MGO were identified as intermediatesof the AGEs using a high-performance liquid chromatographyultraviolet (HPLC-UV) system, as reported previously 16).
Samples were prepared by adding 50 μL aliquots of thereaction mixtures to 6 μL of 5 μg/mL 2,3-butanedione, which
was used as an internal standard, and 75 μL of distilled water.
The mixture was vortexed, following which 125 μL of 6.0 %perchloric acid was added. Then, the mixture was centrifuged
at 12,000 × g for 10 min. The supernatant was added to 125
μL of saturated sodium hydrogen carbonate and 12.5 μL of
1 mg/mL 2, 3-diaminonaphthalene labeling reagent. The
mixture was incubated overnight at 4 °C. The next day, ethyl
acetate (0.5 mL) was added to the reaction solution for liquidliquid extraction, followed by centrifugation at 1,000 × g for
10 min. The supernatant was concentrated by distillation
under a nitrogen atmosphere. Subsequently, 100 μL of MeOH
was added, and the mixture was used as the sample foranalysis. The standards for 3-DG, GO, and MGO wereanalyzed by HPLC using an Inertsil ODS-3 column (5 μm,
150 mm × 4.6 mm i.d., GL Sciences). The mobile phase forgradient elution was as follows: solvent A: H 2 O, solvent B:
100 % acetonitrile. The gradient conditions were as follows:
0 min, 10 % B; 55 min, 41.4% B. The flow rate and detection
wavelength were 1.0 mL/min and 268 nm, respectively.

Statistical analysis

All statistical analyses were performed using GraphPad
Prism 5 for Windows (GraphPad Software, San Diego, CA).
Values are expressed as mean ± standard deviation (SD).
Statistical analysis was performed using one-way ANOVA
followed by Tukey’s test for statistical comparisons amonggroups, with a value of p < 0.05 indicating significance.

Total polyphenol and flavonoid contents
The total polyphenol and flavonoid contents were 214.5± 8.0 mg gallic acid equivalents/g extract and 14 6.6 ± 4.6 mg
catechin equivalents/g extract respectively. The results
indicated that approximately 70 % of the polyphenols in the
crude holy basil extract were flavonoids.

Antioxidant activity

Antioxidant properties of the crude extract are listed inTable 1. The crude extract showed DPPH radical scavenging
activity, ABTS radical cation scavenging activity, and SODlike activity, with IC 50 values of 36.6 ± 0.2 μg/mL, 172.1± 6.5 μg/mL, and 39. 5 ± 3.6 μg/mL, respectively. The IC 50
values of the crude extract obtained in the DPPH radical and
ABTS radical cation scavenging assays were approximately
30 % lower than those of ascorbic acid; However, the values
were approximately 4. 5-fold higher in SOD-like activity.

Structure analysis of isolated compound
The crude extract of holy basil was fractionated bychromatography on DIAION HP-20 column and Sephadex
LH-20 column, followed by purification by preparative HPLC
to isolate phenolic compounds. Luteolin 5-O-glucoside (1),luteolin 7-O-glucuronide (2), (−)-rabdosiin (3), apigenin 7-Oglucuronide (4), rosmarinic acid (5), globoidnan A (6), andsalvianolic acid A (7) were isolated (Fig. 2). Their compounds
were identified by comparison of the data to the references
17- 23). The spectroscopic data of isolated compounds were in
the Supplementary data. Although compounds 1, 2, 3, 4, and5 have been found in various plants, compounds 6 and 7 inholy basil is being reported for the first time.

Inhibitory activity on the formation offluorescent AGEs

The inhibitory activities of the crude extract of holybasil and the isolated compound for the formation offluorescent AGEs are shown in Table 2. Both the crude
extract and isolated compounds inhibited the formation offluorescent AGEs. Their inhibitory activity was stronger
than that of aminoguanidine, which was used as a positive
control. Compounds 3, 5, 6, and 7 significantly suppressed
the formation of fluorescent AGEs.

Table 1. Antioxidant activity of the crude extract of holy basil.
DPPH radical scavenging activity(IC 50 ; μg/mL)

ABTS radical cation scavenging activity(IC 50 ; μg/mL)

SOD-like activity(IC 50 ; μg/mL)

Crude extract

36.6 ± 0. 2 c

172.1 ± 6. 5 c

39.5 ± 3.6 c

Gallic acid

3. 5 ± 0.6 a

12. 4 ± 0.2 a

0. 6 ± 0. 3 a

Ascorbic acid11.8 ± 1.0 b

61.8 ± 9.5 b

179.0 ± 1. 9 b

SampleValues represent mean ± SD from three independent experiments, and means within columns with the same superscript letters are not significantly different( p < 0.05). DPPH,1,1- diphenyl-2-picrylhydrazyl; ABTS, 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); SOD, superoxide dismutase; IC 50, 50 %inhibitory concentration; SD, standard deviation.

_ 179 _

Antiglycative-active Substances of Holy BasilFig. 2. HPLC
chromatogramof the phenolic
compounds
isolatedisolated
from thecrudeof holy basil.
Fig. 2. HPLC
chromatogramof the phenolic
compounds
from
theextract
crude extractof holy basil.

16

Detection: PDA detector (280 nm); gradient condition: 5% ~ 100 % acetonitrile containing 0.1% formic acid.
Detection:
PDA detector(1),(280nm); gradientcondition:~ 100% acetonitrile
containing
0.1% formic acid. 1:
1:
Luteolin 5-O-glucoside
2: Luteolin
7-O-glucronide(2),5%
3: (-)-Rabdosiin(3), 4: Apigenin
7-O-glucronide(4), 5: Rosmarinic acid (5), 6: Globoidnan A (6), 7: Salvianolic acid A (7). HPLC, high-performance liquid
Luteolin 5-O-glucoside(1), 2: Luteolin
7-O-glucronide (2), 3: (-)-Rabdosiin (3), 4: Apigenin 7-O-glucronide (4), 5:
chromatography;
PDA, photodiodearray.

Rosmarinic acid (5), 6: Globoidnan A (6), 7: Salvianolic acid A (7). HPLC, high-performance liquid
chromatography; PDA, photodiode array.

Supplementary data
Spectroscopic data of compounds 1– 7

Luteolin 5-O-glucoside (1): yellow amorphous powder; UV (MeOH) λ max = 248, 346 nm; HR-ESI-MS m/z 447.0945 [MH] -, calcd. for C 21H20 O11, 448.1018; 1H NMR (600 MHz, MeOD): δ 7.37 (dd, 1H, J = 7.6, 2.0 Hz, H-6'), 7.35 (brs, 1H,H-2'), 6.89 (d, 1H, J = 8.2 Hz, H-5'), 6.81 (d, 1H, J = 2.0 Hz, H-6), 6.69 (d, 1H, J = 2.0 Hz, H-8), 6.52 (s, 1H, H-3), 4.83 (d,1H, J = 7.6 Hz, H-1''), 3.94 (dd, 1H, J = 12.0, 1.4 Hz, H-6''α), 3.75 (dd, 1H, J = 12.0, 5.5 Hz, H-6''β), 3.58 – 3.60 (m, 1H,
H-2''), 3.42 – 3.50 (m, 3H, H-3'', H-4'', H-5''); 13 C NMR (125 MHz, MeOD): δ 180.4 (C-4), 165.3 (C-7), 164.4 (C-2), 160.7(C-9), 160.1 (C-5), 150.8 (C-4'), 147.0 (C-3'), 123.5 (C-1'), 120.1 (C-6'), 116.8 (C-5'), 114.0 (C-2'), 109.2 (C-10), 106.5(C-3), 105.1 (C-1''), 104.9 (C-6), 99.3 (C-8), 78.6 (C-5''), 77.3 (C-3''), 74.7 (C-2''), 71.2 (C-4''), 62.5 (C-6''). The above
data were in consistency with the reported values of Luteolin 5-O-glucoside (Sevindik et al., 2015).
_ 18 0 _Glycative Stress Research

Luteolin 7-O-glucronide (2): yellow amorphous powder; UV (MeOH) λ max = 253, 266, 347 nm; HR-ESI-MS m/z 461.0729[M-H]-, calcd. for C 21H18O12, 462.0798; 1 H NMR (600 MHz, MeOD): δ 7.43 (d, 1H, J = 7.6 Hz, H-6'), 7.41 (s, 1H,H-2'), 6.90 (d, 1H, J = 8.3 Hz, H-5'), 6.82 (d, 1H, J = 2.1 Hz, H-8), 6.60 (s, 1H, H-3), 6.49 (d, 1H, J = 2.1 Hz, H-6), 5.10
(d, 1H, J = 7.6 Hz, H-1''), 3.93 (d, 1H, J = 9.0 Hz, H-5''), 3.53 – 3.57 (m, 3H, H-2'', H-3'', H-4''); 13 C NMR (125 MHz,
MeOD): δ 184.1 (C-3), 175.0 (C-6''), 166.9 (C-2), 164.9 (C-7), 162.8 (C-5), 159.0 (C-9), 151.2 (C- 4'), 147.1 (C-3'),
123.5 (C-1'), 120.5 (C-6'), 116.8 (C-5'), 114.3 (C-2'), 107.2 (C-10), 104.1 (C-3), 101.6 (C-1''), 101.4 (C- 6), 96.1 (C8), 77.5 (C-3''), 76.6 (C-5''), 74. 5 (C-2''), 73.3 (C- 4''). The above data were in consistency with the reported values ofluteolin 7-O-glucronide (Bhatarrai et al., 2019).
(−)-Rabdosiin (3): light brown amorphous powder; UV (MeOH) λ max = 255, 285, 345 nm; [α]D = -37.6 (c = 0.50, MeOH),
HR-ESI-MS m/z 717.1475 [M-H]-, calcd. for C 36 H 30 O 16, 718.1549; 1 H NMR (600 MHz, MeOD): δ 7.58 (s, 1H, H-4),
6.79 (s, 1H, H-5), 6.74 (d, 1H, J = 2.1 Hz, H-2''), 6.68 (d, 1H, J = 1.4 Hz, H-2'''), 6.66 (d, 1H, J = 8.2 Hz, H-5'''), 6.64 (d,1H, J = 7.6 Hz, H-5''), 6.60 (d, 1H, J = 8.2 Hz, H-5'), 6.55 (d, 1H, J = 7.6 Hz, H-6''), 6.50 (d, 1H, J = 8.0 Hz, H-6'''), 6.49
(s, 1H, H-8), 6.35 (d, 1H, J = 8.2, 2.0 Hz, H-6'), 6.33 (d, 1H, J = 2.0 Hz, H-2'), 5.04 (dd, 1H, J = 7.9, 4.8 Hz, H-8'''), 4.97
(dd, 1H, J = 6.9, 4.8 Hz, H-8''), 4.44 (brs, 1H, H-1), 3.88 (d, 1H, J = 1.5 Hz, H-2), 3.03 (dd, 1H, J = 14.5, 4.1 Hz, H-7'''),
2.98 (dd, 1H, J = 13.8, 3.8 Hz, H-7'''), 2.91–2.96 (m, 2H, H-7''); 13 C NMR (125 MHz, MeOD): δ 174.3 (C-9'''), 173.7(2-COO-), 173. 5 (C-9'') , 168.0 (3-COO-), 149.1 (C-7), 146.1 (C-3''), 146.0 (C-3'''), 145.9 (C-4'''), 145.4 (C-6), 145.1(C-4''), 145.1 (C-3'), 144.8 (C-4'), 140.9 (C-4), 136.8 (C-1'), 131.4 (C-9), 129.6 (C-1''), 129.5 (C-1'''), 124.7 (C-10),
122.0 (C-6'''), 121.4 (C-6''), 120.5 (C-3), 119.9 (C-6'), 117.7 (C-2'''), 117.6 (C-2''), 117.5 (C-8), 117.3 (C-5), 116.4 (C5''), 116.3 (C-5'''), 116.3 (C-5'), 115.6 (C-2'), 75.7 (C-8''), 75.7 (C-8'''), 49.9 (C-2), 46.1 (C-1), 38.1 (C-7''), 37.7 (C7'''). The above data were in consistency with the reported values of (−)-rabdosiin (Ozgen et al., 2010).
Apigenin 7-O-glucronide (4): yellow amorphous powder; UV (MeOH) λ max = 235, 265, 340 nm; HR-ESI-MS m/z 445.0765[M-H]-, calcd. for C21 H18 O11 , 446.0841; 1 H NMR (600 MHz, MeOD): δ 7.87 (d, 2H, J = 7.6 Hz, H-2', H-6'), 6.92 (d, 2H,
J = 8. 3 Hz, H-3', H-5'), 6.81 (d, 1H, J = 1.8 Hz, H-8), 6.64 (s, 1H, H-3), 6.48 (d, 1H, J = 1.4 Hz, H-6), 5.13 (d, 1H, J = 7.6
Hz, H-1''), 4.03 (d, 1H, J = 8.3 Hz, H-5''), 3.53 – 3.60 (m, 3H, H-2'', H-3'', H- 4''); 13 C NMR (125 MHz, MeOD): δ 184.1(C-4), 175.6 (C- 6''), 166.8 (C-2), 164.6 (C-7), 162.9 (C-5), 162.9 (C-4'), 159.0 (C-9), 129.7 (C-2'), 129.7 (C-6'), 123.1(C-1'), 117.0 (C-3'), 117.0 (C-5'), 107.2 (C-10), 104.1 (C-3), 101.4 (C-1''), 101.2 (C-6), 96.0 (C-8), 77.3 (C-3''), 76.6 (C5''), 74.4 (C-2''), 73.1 (C-4''). The above data were in consistency with the reported values of apigenin 7-O-glucronide(Moussaoui et al., 2010).
Rosmarinic acid (5): brown amorphous solid; UV (MeOH) λ max = 235 and 330 nm; HR-ESI-MS m/z 359.0776 [M-H], calcd. for C18 H16 O8 , 360.0845; 1 H NMR (600 MHz, MeOD): δ 7.54 (d, 1H, J = 15.8 Hz, H-7), 7.03 (d, 1H, J = 2.1 Hz,
H-2), 6.95 (dd, 1H, J = 8.3, 2.0 Hz, H-6), 6.77 (d, 1H, J = 7.6 Hz, H-5), 6.74 (d, 1H, J = 1.5 Hz, H-2'), 6.68 (d, 1H, J = 7.6
Hz, H-5'), 6.61 (dd, 1H, J = 8.2, 2.1 Hz, H-6'), 6.26 (d, 1H, J = 16.5 Hz, H-8), 5.17 (dd, 1H, J = 8.3, 4.1 Hz, H-8'), 3.09 (dd,
1H, J = 14.5, 4.1 Hz, H-7'α), 2.99 (dd, 1H, J = 14.5, 8.3 Hz, H-7'β); 13 C NMR (125 MHz, MeOD): δ 173.7 (C-9'), 168. 5(C-9), 149.7 (C- 4), 147.7 (C-7), 146.8 (C-3), 146.1 (C-3'), 145.2 (C-4'), 129.4 (C-1'), 127.2 (C-1), 123.1 (C-6), 121.8(C-6'), 117.6 (C-2'), 116. 5 (C-5), 116.3 (C-5'), 115.2 (C-2), 114.5 (C-8), 74.8 (C- 8'), 37.9 (C-7'). The above data were inconsistency with the reported values of rosmarinic acid (Sevindik et al., 2015).
Globoidan A (6). light brown amorphous powder; UV (MeOH) λ max = 261 and 318 nm; HR-ESI-MS m/z 491.0987 [M-H]-,
calcd. for C 26 H 20 O10 , 492.1062; 1 H NMR (600 MHz, MeOD): δ 8.24 (brs, 1H, H-4), 7.62 (d, 1H, J = 1.4 Hz, H-2), 7.26 (s,1H, H-8), 7.24 (s, 1H, H-5), 6.87 (d, 1H, J = 8.3 Hz, H-5'), 6.86 (d, 1H, J = 2.0 Hz, H-2'), 6.79 (d, 1H, J = 2.0 Hz, H-2''),6.75 (dd, 1H, J = 8.2, 2.0 Hz, H-6'), 6.68 (dd, 1H, J = 8.2, 2.0 Hz, H-6''), 6.65 (d, 1H, J = 8.2 Hz, H-5''), 5.24 (dd, 1H, J= 9.6, 3.4 Hz, H-8''), 3.16 (dd, 1H, J = 13.4, 3.4 Hz, H-7''), 3.05 (dd, 1H, J = 14.5, 9.6 Hz, H-7''); 13 C NMR (125 MHz,
MeOD): δ 174.2 (C-9''), 168.4 (C-9), 150.2 (C-7), 148.3 (C-6), 146.2 (C-4'), 146.1 (C-3'), 145.9 ( C-3''), 145.2 (C- 4''),140.0 (C-1), 133.8 (C-1'), 131.9 (C-8a), 130.3 (C-1''), 129.8 (C- 4a), 129.4 (C-4), 125.1 (C-3), 124.4 (C-2), 122.4 (C- 6'),121.9 (C- 6''), 118.0 (C-2'), 117.6 (C-2''), 116.4 (C-5''), 116.3 (C-5'), 112.4 (C-5), 109.4 (C-8), 76.0 (C-8''), 38.2 (C-7'').
The above data were in consistency with the reported values of globoidan A (D’Urso et al).
_ 181 _

Antiglycative-active Substances of Holy Basil

Salvianolic acid A (7): yellow amorphous powder; UV (MeOH) λ ma = 227 and 287 nm; HR-ESI-MS m/z 493.1147 [M-H] -,calcd. for C26 H22 O10 , 494.1220; 1 H NMR (600 MHz, MeOD): δ 8.02 (d, 1H, J = 15.8 Hz, H-7'), 7.12 (d, 1H, J = 16.5 Hz,H-8), 7.10 (d, 1H, J = 8.9 Hz, H-6'), 7.03 (d, 1H, J = 2.0 Hz, H-2), 6.86 (dd, 1H, J = 8.3, 2.0 Hz, H-6), 6.74 (d, 1H, J =7.6 Hz, H-5'), 6.73 (d, 1H, J = 7.6 Hz, H-5), 6.71 (d, 1H, J = 2.1 Hz, H-2''), 6.65 (d, 1H, J = 15.8 Hz, H-7), 6.62 (d, 1H, J= 8.3 Hz, H-5''), 6.54 (dd, 1H, J = 8.3, 1.4 Hz, H-6''), 6.29 (d, 1H, J = 15.8 Hz, H-8'), 5.12 (dd, 1H, J = 8.3, 2.7 Hz, H-8''),3.06 (dd, 1H, J = 14. 5, 3.4 Hz, H-7''), 2.92 (dd, 1H, J = 13.8, 9.0 Hz, H-7''); 13 C NMR (125 MHz, MeOD): δ 175. 5 (C-9''),168.9 (C-9'), 148.2 (C- 4'), 146.9 (C-7'), 146.7 (C-4), 146.4 (C-3), 146.0 (C-3''), 145.0 (C-4''), 144.4 (C-3'), 137.8 (C7), 131.4 (C-1), 130.0 (C-1''), 128.2 (C-2'), 126.2 (C-1'), 122.0 (C-6''), 120.6 (C-8), 120.4 (C-6), 120.1 (C-6'), 117.3 (C2''), 116. 5 (C-5''), 116.3 (C-5), 116.1 (C-8'), 114.7 (C-5'), 114.0 (C-2), 76.3 (C-8''), 38.2 (C-7''). The above data were inconsistency with the reported values of salvianolic acid A (Zhou et al., 2014).

Table 2. Inhibitory activity against fluorescent AGE formation.
IC 50 value (mg/mL)

IC 50 value (mM)

Crude extract

0.37 ± 0.02 b-

Luteolin 5-O-glucoside (1)0.22 ± 0.02 c

0.49 ± 0.05 b

SampleLuteolin 7-O-glucronide (2)(-)-Rabdosiin (3)Apigenin 7-O-glucronide (4)Rosmarinic acid (5)Globoidnan A (6)Salvianolic acid A (7)Aminoguanidine

0.25 ± 0.01 c

0. 55 ± 0.01 b

0.23 ± 0.01 c

0. 52 ± 0.02 b

0.15 ± 0.01d

0. 21 ± 0.02 c

0.07 ± 0.01 e

0.20 ± 0.04 c

0.09 ± 0.01 e

0.18 ± 0.03 c

0.08 ± 0.01 e

0.49 ± 0.04 a

0.16 ± 0.02 c

6.74 ± 0.58 a

Values represent mean ± SD from three independent experiments, and means within columns with the same superscript
letters are not significantly different ( p < 0.05). AGE, advanced glycation end product; SD, standard deviation.

Inhibitory activity on the formation of CML and
AGE intermediates

To examine the inhibitory activity for the formation of
non-fluorescent AGEs, the level of CML, which is one of theAGEs, was measured using ELISA. As shown in Fig. 3, the
CML levels decreased in the crude extract of holy basil andthe compounds isolated from it. The inhibition rates of CML
formation in the crude extract, compounds 1, 2, and 4 were
10 % ~ 20 % lower than that of the control. Compounds 3, 5,
6, and 7 were more potent in inhibiting CML formation thanthat of the crude extract, compounds 1, 2, and 4.
Next, we evaluated the effects of the crude extract ofholy basil and the compounds isolated from it on theformation of AGE intermediates (Fig. 4 ). HPLC-UV analysis
revealed the formation of three types of intermediates,
namely, 3-DG, GO, and MGO. Both crude extract and theisolated compounds suppressed the formation of 3-DG,
GO, and MGO. In particular, compounds 3, 5, 6, and 7
significantly reduced the formation of AGE intermediates.
The crude extract showed approximately 30 % inhibitory

activity against 3-DG formation; compounds 1, 2, and 4
showed approximately 50 % activity; and compounds 3, 5, 6,
and 7 showed the highest inhibition activities (approximately
70 %). The formation of GO and MGO was inhibited by 40 %~ 50 % upon the addition of the crude extract, compounds 1,
2, and 4; and compounds 3, 5, 6, and 7 had potent inhibitory
activity of AGE intermediates (approximately 60% ~ 70%).

Discussion
Glycation stress in vivo is one of the causes of aging andlifestyle-related diseases, and AGE formation by glycation isassociated with oxidative reactions. Aminoguanidine inhibits
AGE production; however, ingestion of the compound causes
side effects such as anemia, liver damage, and vitamin B6
deficiency 24). Therefore, new antiglycation components, suchas luteolin, cyanidin 3-O-galactoside, and rhodanthenone B
have been isolated from medicinal herbs 25-27).
Holy basil is an important herb in traditional medicines_ 18 2 _Glycative Stress Research

Percentage of CML formation

150

a
100

b

b

b

b

b

d

c

cd

e

50

(6
)

(7

ne

A

A

i di

n

id

an
gu

Am

in o

ac
c

oli

an

Sa

lvi

ob

oi d

na

)

(5
)
id

(4
)

sm

ar

ini

c

ac

on

ur

luc

-g

Ro

Gl

n

i de

(3
)

(2
)

s ii

do
ab

Ap

Lu

te

i ge

oli

nin

7O

()

-g

-R

luc

ur

on

os

i de

ac

luc
-g

O

7O

n

5n
oli
te
Lu

i de

t

l
ro

ex
tr

nt

Cr

ud

e

Co

(1
)

0

Fig. 3. Effects of the crude extract of holy basil and the phenolic compounds isolated from it on theformation of CML.

Extract and isolated compounds with indicated percentages were introduced into the BSA-glucose model andincubated at 60 °C for seven days. CML was quantified using ELISA. Data were calculated as the percentage of
CML formation compared to that of controls without sample addition and are presented as mean ± SD (n = 8). Barwith different letters ( a - e) differ ( p < 0.05). CML, N ε-(carboxymethyl) lysine; ELISA, enzyme-linked immuno
sorbent assay; SD, standard deviation.

150

150

150

Co

e

ud

Cr

5-

n

oli

Fig. 4. Effects of the crude extract of holy basil and the phenolic compound isolated from it on the formation of
AGE intermediate.

Extract and isolated compounds with indicated percentages were introduced into the BSA-glucose model and incubatedat 60 °C for seven days. The AGE intermediates were detected using a HPLC-UV system: (A) 3-DG, (B) GO, and (C)MGO. Data were calculated as the percentage of AGE intermediate formation compared to that of controls withoutsample addition and are presented as mean ± SD (n = 3). Different letters ( a - e ) for the same method represent statisticallysignificant differences between samples ( p < 0.05). AGE, advanced glycation end product; BSA, bovine serum albumin;
HPLC, high-performance liquid chromatography; UV, ultra violet; 3-DG, 3- deoxyglucosone; GO, glyoxal; MGO,
methylglyoxal; SD, standard deviation.

_ 18 3 _

c

nt
ro
ex l
O
tra
n
glu
ct
7O
co
-g
luc si d
Ap
e
u
(
ro
ige
1)
(ni d
nin
)-R
e
ab
(2
7)
do
O
-g
luc siin
(3
Ro
ur
)
on
sm
ide
ar
ini
(4
c
)
Gl
ac
ob
id
Sa
oid
(
5)
lvi
n
a
an
n
oli
A
c
(6
)
A m ac i d
A
in o
(7
gu
)
an
idi
ne

Lu

b
c

0

te

n
oli
te

c

c

oli

c

b

b

te

c

0

Lu

n

n

oli

te

oli

te

Lu

Lu

c

b

50

Lu

-g

O

c

5-

Cr

ud

e

Co

nt
ro
ex l
tra
ct
7luc
O
o
-g
luc si d
Ap
e
u
(
ro
ige
1)
(ni d
nin
)-R
e
ab
(2
7)
do
O
-g
luc siin
(3
Ro
ur
)
on
sm
ide
ar
ini
(4
c
)
Gl
a
ob
Sa
oid c id (
5)
lvi
na
an
n
oli
A
c
(6
)
A m ac i d
A
in o
(7
gu
)
an
idi
ne

0

50

b

Lu

e

b

e
5ex l
O
tra
-g
ct
7luc
O
os
-g
luc
ide
Ap
ur
ige
on (1 )
(nin
)-R
ide
ab
(2
7)
do
O
-g
luc siin
(3
Ro
ur
)
on
sm
ide
ar
ini
(4
c
)
Gl
ac
ob
id
Sa
oid
(5
lvi
n
)
an
an
oli
A
c
(6
)
A m ac i d
A
in o
(7
gu
)
an
idi
ne

e

n

e

b

b

oli

e

b

te

d

b

ro

50

c

cd

ud

cd

a

100

nt

b

100

Co

100

a

Cr

a

Percentage of MGO formation(C )Percentage of GO formation( B)

Percentage of 3-DG formation(A)Antiglycative-active Substances of Holy Basiland is often referred to as "The Queen of Herbs" or "The
Incomparable One" or "The Mother Medicine of Nature
in Ayurveda Systems" 28). This herb has a history of morethan 3000 years as a medicinal plant in India and Sri
Lanka and has potential functional properties. In this study,
the physiological functions of holy basil were evaluatedusing its antiglycative effect as an indicator. The hot water
extract of holy basil exhibited strong antiglycation activity.
The extract was mostly composed of flavonoids such asluteolin 5-O-glucoside (1), luteolin 7-O-glucuronide (2),(−)-rabdosiin (3), apigenin 7-O-glucronide (4), rosmarinicacid (5), globoidnan A (6), and salvianolic acid A (7), andthese flavonoids were found to be active components for theantiglycative effect. In particular, compounds 3, 5, 6, and 7
showed stronger antiglycation activities than glycosides.
Yang et al. 29) reported that kaempferol exhibited stronger
antiglycation inhibitory activity than kaempferol glycosides
in the BSA-glucose model, which is consistent with our
results. Incidentally, compounds 6 and 7 have been isolated
and identified in holy basil leaves for the first time. The
mechanism of in vivo glycation involves the accumulationof AGEs due to the metabolism of dicarbonyl derivatives
such GO, 3-DG, and MGO 3, 30). Among the AGEs, CML,
which is generated by an intramolecular Cannizzaro reaction
with GO as a precursor and by oxidative cleavage of Schiffbases or Amadori compounds, has been detected in patientswith diabetes and high oxidative stress 31). Therefore, theeffect of the crude extract of holy basil and the compounds
isolated from it on the glycation reaction was examined, andindeed, GO formation was significantly inhibited. This result
suggested that the inhibition of GO formation led to the
suppression of CML accumulation by preventing subsequent
metabolism.
In a previous study, free radical scavenging was
proposed as an anti-glycation mechanism 32). The crude
extract of holy basil scavenged DPPH and ABTS radical
cations and also exhibited SOD-like activity. These results
suggest that the antioxidant properties of holy basil help
in inhibiting AGE formation. Oxidative stress plays animportant role in the mechanism of AGE formation andaccumulation in vivo and is implicated as a key factor in
the progression of several diseases. Glycation, which isaccelerated in the presence of oxidative stress in tissues,
induces aging 33). It has been suggested that the extract ofholy basil, which has potent antioxidant activity, can be aneffective component in inhibiting age-related glycation;
however, further research is needed to elucidate the in vivo
antiglycative mechanism.

intermediates. It has been suggested that the antioxidant
activity of these phenolic compounds can contribute to theantiglycation effect. Our findings showed that compounds
3, 5, 6, and 7 were the active constituents of holy basil. The
properties of holy basil are beneficial for the development ofcosmetics, supplements, medicines, and other applications.

Acknowledgments
The authors acknowledge Ayano Kinjo for supporting
this study. The publication of this study was supported by
the Isyoku-Dogen Research Foundation (IDF#221001).

Conflict of interest
The authors have no conflicts of interest directly
relevant to the content of this article.

Conclusions

Herein, we report the antiglycative effect of the extractof holy basil and the phenolic compounds isolated fromit on the BSA-glucose system. The crude extract of holybasil is rich in polyphenols, and luteolin 5-O-glucoside(1), luteolin 7-O-glucuronide (2), (−)-rabdosiin (3), apigenin
7-O-glucuronide (4), rosmarinic acid (5), globoidnan A (6),and salvianolic acid A (7) present in it significantly inhibitedthe formation of fluorescent AGEs, CML, and glycation_ 18 4 _Glycative Stress Research

References
1) Singh R, Barden A, Mori T, et al. Advanced glycation-

endproducts:a review. Diabetologia. 2001; 44: 129-146.
2) Ichihashi M, Yagi M, Nomoto K, et al. Glycation stress andphoto-aging in skin. Anti-Aging Med. 2011; 8: 23-29.
3) Ahmed N. Advanced glycation end products: Role inpathology of diabetic complications. Diabetes Res Clin
Pract. 2005; 67: 3-21.
4) Takedo A, Yasuda T, Miyata T, et al. Immunohistochemical
study of advanced glycation end products in aging and
Alzheimer’s disease brain. Neurosci Lett. 1996; 221: 17-20.
5) Uribarri J, Cai W, Peppa M, et al. Circulating glycotoxins
and dietary advanced glycation endproducts: Two links
to inflammatory response, oxidative stress, and aging.
J Gerontol A Biol Sci Med Sci. 2007; 62: 427-433.
6) Cohen MM. Tulsi – Ocimum sanctum: A herb for all
reasons. J Ayurveda Integr Med. 2014; 5: 251-259.
7) Kunihiro K, Kikuchi Y, Nojima S, et al. Characteristic ofaroma components and antioxidant activity of essential oilfrom Ocimum tenuiflorum leaves. Flavour & Fragrance J.
2022; 37: 210-218.
8) Pattanayak P, Behera P, Das D, et al. Ocimum sanctum
Linn. A reservoir plant for therapeutic applications: An
overview. Pharmacogn Rev. 2010; 4: 95-105.
9) Zhao L, Zhu X, Yu Y, et al. Comprehensive analysis of theanti-glycation effect of peanut skin extract. Food Chem.
2021; 362: 130169.
10) Venuprasad MP, Kandikattu HK, Razack S, et al.
Chemical composition of Ocimum sanctum by LC-ESIMS/MS analysis and its protective effects against smoke
induced lung and neuronal tissue damage in rats. Biomed
Pharmacother. 2017; 91: 1-12.
11) Kusindarta DL, Wihadmadyatami H, Haryanto A. The
analysis of hippocampus neuronal density (CA1 and CA3)
after Ocimum sanctum ethanolic extract treatment on theyoung adulthood and middle age rat model. Vet World.
2018; 11: 135-140.
12) Singleton VL, Orthofer R, Lamuela-Raventós RM. [14]Analysis of total phenols and other oxidation substrates
and antioxidants by means of Folin-Ciocalteu reagent.
Methods Enzymol. 1999; 299: 152-178.
13) Yao X, Zhu L, Chen Y, et al. In vivo and in vitro antioxidant
activity and α-glucosidase, α-amylase inhibitory effectsof flavonoids from Cichorium glandulosum seeds. Food
Chem. 2013; 139: 59-66.
14) Hori M, Yagi M, Nomoto K, et al. Inhibition of advanced
glycation end product formation by herbal teas and its
relation to anti-skin aging. Anti-Aging Med. 2012; 9: 135148.
15) Shin S, Son D, Kim M, et al. Ameliorating effect of Akebia
quinata fruit extracts on skin aging induced by advanced
glycation end products. Nutrients. 2015; 7: 9337-9352.
16) Takabe W, Yagi M, Ichihashi M, et al. Anti-glycative
effect of palladium and platinum nanoparticle solution.
Glycative Stress Res. 2016; 3: 222-228.
17) Sevindik HG, Ozgen U, Atila A, et al. Phtytochemical
studies and quantitative HPLC analysis of rosmarinic acidand luteolin 5-O-β-D-glucopyranoside on Thymus praecox
subsp. grossheimii var. grossheimii. Chem Pharm Bull
(Tokyo). 2015; 63: 720-725.

18) Bhatarrai G, Seong SH, Jung HA, et al. Isolation andquantitative analysis of BACE1 inhibitory compounds from
Cirsium maackii flower. Nat Prod Sci. 2019; 25: 326-333.
19) Ozgen U, Sevindik H, Kazaz C, et al. A new sulfated alphaionone glycoside from Sonchus erzincanicus Matthews.
Molecules. 2010; 15: 2593-2599.
20) Flegkas A, Milosević Ifantis T, Barda C, et al.
Antiproliferative activity of (−)-rabdosiin isolated from
Ocimum sanctum L. Medicines (Basel). 2019; 6: 37.
21) Moussaoui F, Zellagui A, Segueni N, et al. Flavonoid
constituents from Algerian Launaea resedifolia (O.K.) andtheir antimicrobial activity. Rec Nat Prod. 2010; 4: 91-95.
22) D’Urso G, Masullo M, Seigner J, et al. LC-ESI-FT-MSn
metabolite profiling of Symphytum officinale L. roots leads
to isolation of comfreyn A, an unusual arylnaphthalenelignan. Int J Mol Sci. 2020; 21: 4671.
23) Zhou W, Xie H, Xu X, et al. Phenolic constituentsfrom Isodon lophanthoides var. graciliflorus and theirantioxidant and antibacterial activities. J Funct Foods.
2014; 6: 492-498.
24) Yagi M, Sakiyama C, Miyata Y, et al. Antiglycative effectof genipin and crocetin. Glycative Stress Res. 2021; 8:
156-161.
25) Yagi M, Nomoto K, Hori M, et al. The effect of edible
purple Chrysanthemum extract on advanced glycation
end products generation in skin: A randomized controlled
clinical trial and in vitro study. Anti- Aging Med. 2012; 9:61-74.
26) Takabe W, Yamaguchi T, Hayashi H, et al. Identificationof antiglycative compounds in Japanese red water pepper(red leaf variant of the Persicaria hydropiper sprout).
Molecules. 2018; 23: 2319.
27) Ohno R, Moroishi N, Sugawa H, et al. Mangosteen
pericarp extract inhibits the formation of pentosidine andameliorates skin elasticity. J Clin Biochem Nutr. 2015; 57:
27-32.
28) Rastogi S, Kalra A, Gupta V, et al. Unravelling the genomeof Holy basil: An “incomparable” “elixir of life” of
traditional Indian medicine. BMC Genomics. 2015; 16: 413.
29) Yang R, Wang WX, Chen HJ, et al. The inhibition ofadvanced glycation end-products by five fractions andthree main flavonoids from Camellia nitidissima Chi
flowers. J Food Drug Anal. 2018; 26: 252-259.
30) Rabbani N, Thornalley PJ. Dicarbonyl stress in cell andtissue dysfunction contributing to ageing and disease.
Biochem Biophys Res Commun. 2015; 458: 221-226.
31) Takeuchi M, Makita Z. Alternative routes for theformation of immunochemically distinct advanced
glycation end-products in vivo. Curr Mol Med. 2001; 1:
305-315.
32) Wu CH, Huang SM, Lin JA, et al. Inhibition of advanced
glycation endproduct formation by foodstuffs. Food Funct.
2011; 2: 224-234.
33) Kim CS, Park S, Kim J, et al. The role of glycation in thepathogenesis of aging and its prevention through herbal
products and physical exercise. J Exerc Nutrition Biochem.
2017; 21: 55-61.

_ 18 5 _