Synthesis and evaluation of novel ascorbyl cinnamates as potential anti-oxidant and antimicrobial agents

Article abstract of DOI:10.1007/s11164-017-2970-z

Abstract: An efficient preparation procedure has been proposed for the synthesis of 5,6-di-O-cinnamyl-l-ascorbic acid ester (5,6-CA–AA) and 2-O-cinnamyl-l-ascorbic acid ester (2-CA–AA) via ascorbic acid (AA) and cinnamic acid (CA), and their anti-oxidant and antibacterial activities were also investigated. Among the factors affecting the synthesis efficiency of 5,6-CA–AA and 2-CA–AA, reaction solvent and substrate molar ratio were very important, resulting in 76 and 54% total yield, respectively. Further studies indicated that 5,6-CA–AA maintained the inherent inhibitory ability of AA in α, α-diphenyl-β-picrylhydrazyl radical scavenging assay. Also, it had stronger scavenging activity against hydroxyl radicals than that of standard tert-butylhydroquinone (EC₅₀: 7.84 vs. 8.38?mM). In addition, 5,6-CA–AA and 2-CA–AA possessed comparable antibacterial activity to that of CA. Significantly, the application of ascorbyl cinnamate could retard the lipid oxidation of peanut oil and pork, and the corresponding perioxide value and thiobarbituric acid reactive substance content decreased by 32.0 and 56.5%, respectively. Moreover, the microbial reproduction of the pork treated with mixed ascorbyl cinnamate was attenuated to some extent. These investigations will pave the way for designing more efficient bifunctional derivatives of AA and CA.

Full text of DOI:10.1007/s11164-017-2970-z

Res Chem Intermed
DOI 10.1007/s11164-017-2970-z
Synthesis and evaluation of novel ascorbyl cinnamates
as potential anti-oxidant and antimicrobial agents
Ailing Hui1 Yuan Wang1 Kaili Zhu1
•
•
•
Jingchao Chen1 Qingping Fan1 An Zhou1,2
Wencheng Zhang1 Zeyu Wu1
•
•
•
•
Received: 7 November 2016 / Accepted: 2 May 2017
Ó Springer Science+Business Media Dordrecht 2017
Abstract An efficient preparation procedure has been proposed for the synthesis of
5,6-di-O-cinnamyl-L-ascorbic acid ester (5,6-CA–AA) and 2-O-cinnamyl-L-ascorbic
acid ester (2-CA–AA) via ascorbic acid (AA) and cinnamic acid (CA), and their anti-
oxidant and antibacterial activities were also investigated. Among the factors
affecting the synthesis efficiency of 5,6-CA–AA and 2-CA–AA, reaction solvent and
substrate molar ratio were very important, resulting in 76 and 54% total yield,
respectively. Further studies indicated that 5,6-CA–AA maintained the inherent
inhibitory ability of AA in a, a-diphenyl-b-picrylhydrazyl radical scavenging assay.
Also, it had stronger scavenging activity against hydroxyl radicals than that of
standard tert-butylhydroquinone (EC50: 7.84 vs. 8.38 mM). In addition, 5,6-CA–AA
and 2-CA–AA possessed comparable antibacterial activity to that of CA. Signifi-
cantly, the application of ascorbyl cinnamate could retard the lipid oxidation of
peanut oil and pork, and the corresponding perioxide value and thiobarbituric acid
reactive substance content decreased by 32.0 and 56.5%, respectively. Moreover, the
microbial reproduction of the pork treated with mixed ascorbyl cinnamate was
attenuated to some extent. These investigations will pave the way for designing more
efficient bifunctional derivatives of AA and CA.
&
Ailing Hui
haling@hfut.edu.cn
1
2
Engineering Research Center of Bio-process, Ministry of Education, Hefei University of
Technology, 134 Mailbox, No. 193 Tunxi Road, Hefei 230009, Anhui, China
Anhui Province Key Laboratory of Research and Development of Chinese Medicine, Anhui
University of Traditional Chinese Medicine, Hefei 230038, Anhui, China
123

A. Hui et al.
Graphical Abstract
O a
a
n
n
t
t
HO
O
i
i
m
o
x
i
O
c
i
d
r
HO H
HO
o
a
+
OH
b
n
i
t
OH
a
l
Cinnamic acid (CA)
Ascorbic acid (AA)
bifunctional
compound
O
O
O
O
O
O
O
O
C
HO
O
H
O
OH
HO
HO
HO
7 (2-CA-AA)
18 (5,6-CA-AA)
antioxidant ?
antimicrobial?
DPPH·, ·OH
Inhibition zone
Microbial growth,fat oxidation
Lipid oxidation
Keywords Ascorbic acid Á Cinnamic acid Á Anti-oxidant activity Á Antibacterial
activity Á Lipid oxidation
123

Synthesis and evaluation of novel ascorbyl cinnamate as…
Introduction
L-ascorbic acid (AA) is an important anti-oxidant in drug, food and biological
systems [1], but it is easily soluble and unstable under aqueous conditions, which
hinder its application in lipid condition. To overcome this problem, AA has been
chemically modified by protecting alcohol functions or the enediol system. Among
these derivatives, 2-O-a-D-glucopyranosylascorbic acid 1 and salts of ascorbyl
2-phosphoric esters 2 (Fig. 1) have been successfully used in cosmetics and food
additives [2, 3]. Moreover, some active substances were linked at the C-2 or C-3
hydroxyl group of AA so as to demonstrate multiple biological properties. AA/kojic
acid hybrids 3 [4] and AA/ferulic ester hybrids 4–6 [5] were part of them. Also,
Galey developed the preparation of 2-O-ascorbyl cinnamate derivatives 7–10
(Fig. 2) and diesters of AA such as 2-O-[6-palmitoyl ascorbyl]-40-acetoxy ferulate
11, and their protective properties against oxidation of lipid constituents of the skin
have also been claimed [6].
Compared with those modified in the enediol position, the derivatives modified in
the alcohol position of AA have been widely investigated, especially the potential
bifunctional compound. ascorbyl benzoate 12 (Fig. 3) was achieved through lipase-
catalysis in cylcohexanone, and its anti-oxidant capacity was comparable to that of
ascorbyl palmitate, although its antimicrobial activity was weaker than that of
benzoic acid [7, 8]. In contrast, ascorbyl cinnamate was of greater interest because
cinnamic acid (CA) is a traditional antimicrobial agent [9, 10]. Thomas reported the
preparation of specific ascorbyl cinnamate 13–16 via direct esterification, enzymatic
esterification or using pre-activated acyl halide and their application as UV filters to
protect skin [11]. 6-O-cinnamyl-L-ascorbic acid ester 17 was prepared by reacting
OH
O
HO
HO
HO
HO
O
6
1
HO
O
5
O
4
O
HO
2
OH
H
O
HO H
H
HO
O
Mg2+
3
OH
OH
OH
HO
O P O
O
O
O
L-ascorbic acid
2
1
HO
HO
HO
HO
O
O
O
O
H
O
H
HO
OH
O
O
4 R = CH3
5 R = CH2CH3
6 R = (CH2)11CH3
OH
O
O
OH
MeO
CO2R
3
Fig. 1 Ascorbic acid and its novel derivatives modified in the enediol position
123

A. Hui et al.
R4O
HO
O
O
H
7
8
9
R1 = R2 = R3 = R4 = H
R1 = OCH3, R2 = OH, R3 = R4 = H
R1 = OH, R2 = OH, R3 = R4 = H
O
HO
O
10 R1 = R3 = OCH3, R2 = OH, R4 = H
11 R1 = R2 = OCH3, R3 = H, R4 = C15H31CO
R3
R1
R2
Fig. 2 2-O-ascorbyl cinnamate and its novel derivatives
OH
HO
OH
HO
O
H
OH
H
OH
13 R1 = R2 = R3 = H
O
O
O
O
14 R 1 = R2 = H, R3 =OCH3
15 R1 = OCH3, R2 = R3 = H
16 R1 = R3 = H, R2 = OCH3
O
O
O
H3CO
R1
R3
R2
12
OCH3
OH
O
O
O
O
OH
HO
17
Fig. 3 Some potential bifunctional AA derivatives modified in alcohol position
AA with vinyl cinnamic acid in the presence of immobilized Candida antarctica
lipase (Novozyme 435), but it only resulted in 68% yield after stirring for 6 days at
30 °C [12]. Shortly afterwards, chemosynthesis of 17 was also realized with 61%
yield. However, it required that AA reacted with cinnamyl chloride for 24 h at 0 °C
[13].
In the present study, 5,6-di-O-cinnamyl-L-ascorbic acid ester 18 (5,6-CA–AA)
was obtained with 76% yield when the reaction was carried out in a mixed solvent
of N, N-dimethylformide (DMF) and dichloromethane (CH2Cl2) at room temper-
ature (RT). Also, an efficient three-step access to 2-O-cinnamyl-L-ascorbic acid
ester 7 (2-CA–AA) with 54% overall yield has been developed (Scheme 1).
Moreover, the stability, anti-oxidant and antimicrobial properties of 5,6-CA–AA
and 2-CA–AA were further assessed.
123

Synthesis and evaluation of novel ascorbyl cinnamate as…
O
O
O
O
O
O
HO
O
+
O
O
H
HO
OH
triethylamine
DMF-CH2Cl2
HO H
HO
OH
Cl
18 (5,6-CA-AA)
HO
HO
O
O
O
Cinnamyl chloride,
triethylamine
Acetone
POCl3
H3C
H3C
O
O
O
OH
Tetrahydrofuran
HO
HO
OH
19
O
O
O
C
O
O
O
O
HO
HCl(aq.)-EtOH
pH = 1
O
C
H3C
H3C
O
O
HO
HO
20
HO
7 (2-CA-AA)
Scheme 1 Synthesis of 5,6-di-O-cinnamyl-L-ascorbic acid ester and 2-O-cinnamyl-L-ascorbic acid
Experimental
L-ascorbic acid, cinnamic acid, thionyl chloride, tert-butylhydroquinone (TBHQ), a,
a-diphenyl-b-picrylhydrazyl (DPPH) and phenanthroline were purchased from
Sinopharm Chemical Reagent (Shanghai, China) or Alfa (Shanghai, China).
Potassium sorbate (PS) was purchased from Nantong Acetic Acid Chemical
(Nantong, China). All the reagents were used without additional purification. All the
solvents and other reagents were of analytical grade. Thin layer chromatography
was used to monitor the formation of the products and their chemical structure was
determined by infrared spectroscopy (IR), nuclear magnetic resonance (NMR)
spectra and electrospray ionization mass spectrometer (ESI–MS). IR spectra were
recorded on a Nicolet 5700 FT-IR spectrophotometer. 1H-NMR (600 MHz) and 13
C
NMR (150 MHz) spectra were recorded on an Agilent VNMRS 600 spectrometer.
Chemical shifts are expressed in parts per million downfield from tetramethylsilane
as an internal standard. ESI–MS analyses were performed on a Finnigan LCQ
Advantage MAX spectrometer.
Escherichia coli ATCC 11775, Bacillus subtilis ATCC 6633 and Staphylococcus
aureus ATCC 6538 were preserved by our laboratory. Nutrient Agar medium was
purchased from Hangzhou Microbial Reagent (Hangzhou, China). Refined,
bleached and deodorized peanut oil was bought from a local market. Shuanghui
chilled pork was bought from Carrefour supermarket in Hefei.
123

A. Hui et al.
Procedure for synthesis of 5,6-di-O-cinnamyl-L-ascorbic acid (5,6-CA–AA)
Cinnamyl chloride was prepared by reacting cinnamic acid (2.96 g, 20 mmol) with
thionyl chloride (12 mL) at 45 °C for 4 h. Then, unreacted thionyl chloride was
removed under vacuum and the desired acyl chloride was diluted with 6 mL CH2Cl2
and stored on standby. To a solution of AA (1.76 g, 10 mmol) in DMF (8 mL) and
CH2Cl2 (6 mL) was added triethylamine (Et3N, 20 mmol) and the mixture was
cooled to 0 °C. Cinnamyl chloride (20 mmol, diluted in 6 mL CH2Cl2) was added
dropwise and the resulting mixture was stirred at RT for 8 h. Water (30 mL) was
poured in to terminate the reaction, then extracted with CH2Cl2 (30 mL), washed
with water, dried with MgSO4 and concentrated in vacuo. The residue was separated
by silica gel chromatography with elution consisting of chloroform/methanol (5:1,
v/v) to afford 3.08 g of 5,6-di-O-cinnamyl-L-ascorbic acid as a olorless foam solid
(yield: 76%). mp 121–123 °C. IR (KBr, cm-1): mmax 3403.6, 3061.1, 3027.9,
2922.8, 2850.8, 1713.6, 1635.8. 1H NMR (600 MHz, CDCl3): d 7.33–7.99 (m, 12H,
Ph and PhCH=), 6.59–6.62 (m, 1H, =CHCO), 6.48–6.49 (m, 1H, =CHCO), 4.95 (d,
1H, J = 1.2 Hz, C4-H), 4.37–4.54 (m, 3H, C5-H, C6-H). 13C NMR (150 MHz,
CDCl3): d 170.1, 166.5, 154.1, 145.2, 135.2, 129.7, 128.1, 126.4, 118.6, 116.9, 74.8,
70.3, 63.1. ESI–MS: m/z 435.12 [M-H]-.
A small amount of 6-O-cinnamyl-L-ascorbic acid 17 was also obtained during the
preparation of 5,6-CA–AA [14]. 17: 1H NMR (600 MHz, CDCl3): d 7.58–7.83 (m,
6H, Ph and PhCH=), 6.42–6.45 (d, 1H, J = 16.2 Hz, =CHCO), 4.55–4.59 (m, 3H,
C4-H, C6-H), 3.90 (m, 1H, C5-H).
Procedure for synthesis of 2-O-cinnamyl-L-ascorbic acid (2-CA–AA)
To a magnetically stirred solution of AA (1.76 g, 10 mmol) in acetone (9 mL),
phosphorus oxychloride (0.5 mL) was added and the reaction mixture was stirred
for 4 h at RT. Then it was filtered, washed with cold acetone–water and dried in
vacuo, leading to 5,6-O-isopropylidene-L-ascorbic acid 19 being obtained as
colorless granules (1.92 g, 89%). IR (KBr, cm-1): mmax 3242.6, 2993.3, 2907.6,
2741.6, 1755.5, 1665.4. 1H NMR (600 MHz, CD3COCD3): d 4.71–4.73 (m, 1H, C4-
H), 4.37–4.39 (m, 1H, C5-H), 4.16–4.21 (m, 1H, C6-H), 3.99–4.03 (m, 1H, C6-H),
1.29 (s, 6H, Me).
The above 5,6-O-isopropylidene-L-ascorbic acid 19 was dissolved in the mixed
solvent of tetrahydrofuran (THF, 20 mL) and CH2Cl2 (8 mL), and then dried Et3N
(13.5 mmol) was added. The reaction mixture was cooled to 0 °C, cinnamyl
chloride (13.5 mmol, 10 mL in CH2Cl2) was added dropwise and the resulting
solution was stirred at RT for another 6 h. When the reaction was terminated, water
(30 mL) and CH2Cl2 (30 mL) was added to the mixture, then extracted, washed
with water, dried with MgSO4 and concentrated in vacuo. The crude product 20 was
obtained without any purification for the next reaction. IR (KBr, cm-1): mmax
3445.6, 3062.6, 2986.9, 2935.6, 1705.0, 1596.6, 1450.7, 1496.9. 1H NMR
(600 MHz, CD3COCD3): d 7.74–7.77 (m, 1H, PhCH=), 7.60–7.66 (m, 2H, Ph),
7.38–7.45 (m, 3H, Ph), 6.56–6.60 (m, 1H, =CHCO), 4.47 (m, 1H,C4-H), 4.33 (m,
123

Synthesis and evaluation of novel ascorbyl cinnamate as…
1H, C5-H), 4.08–4.11 (m, 1H, C6-H), 4.05–4.06 (m, 1H, C6-H), 1.30 (s, 3H, Me),
1.25 (s, 3H, Me).
The above product 20 was added to a HCl solution (pH 1, 12 mL) and EtOH
(8 mL) for de-protection, then the mixture was stirred at RT for 10 h, and
evaporated under vacuum, neutralized with NaHCO3 solution, and extracted with
ethyl acetate. The organic layer was collected and washed with water, dried with
MgSO4 and concentrated in vacuo. The residue was chromatographed on silica gel
with elution solvent consisting of chloroform/methanol (5:1, v/v), and 2-O-
cinnamyl-L-ascorbic acid 7 (1.65 g, 5.4 mmol, 54% total yield) was obtained as a
colorless foam solid. mp 139–141 °C. IR (KBr, cm-1): mmax 3421.1, 1718.0, 3002.0,
2936.3, 1718.0, 1577.2, 1442.1, 1424.7. 1H NMR (600 MHz, CD3COCD3): d
7.77–7.80 (m, 1H, PhCH=), 7.65–7.71 (m, 2H, Ph), 7.40–7.45 (m, 3H, Ph), 6.64 (d,
1H, J = 16.2 Hz, =CHCO), 4.45–4.49 (s, 2H, C5-OH, C6-OH), 4.20–4.27 (d, 1H,
J = 1.2 Hz, C4-H), 3.95–4.04 (m, 1H, C5-H), 3.70–3.82 (m, 2H, C6-H). 13C NMR
(150 MHz, CD3COCD3): d 168.3, 165.3, 158.5, 148.6, 134.9, 128.7, 128.0, 126.5,
115.7, 111.1, 76.8, 68.1, 62.1. ESI–MS: m/z 305.07 [M-H]-.
Stability of 5,6-CA–AA and 2-CA–AA in solution
AA, 5,6-CA–AA (18) and 2-CA–AA (7) was dissolved in ethanol and scanned by
ultraviolet spectrophotometer with full wavelength mode (UV-1700; Ruili, China),
and their maximum wavelengths (kmax) were detected at 245, 284, and 265 nm,
respectively.
The solutions of AA, 5,6-CA–AA and 2-CA–AA in ethanol were warmed to
37 °C for a period of time. The absorbance at kmax was measured at intervals of 1 h.
Then, the solutions of 5,6-CA–AA and 2-CA–AA in ethanol-phosphate buffer
solution (PBS) were stored at RT for several weeks, and the absorbance was
similarly recorded.
Anti-oxidant property of 5,6-CA–AA and 2-CA–AA
DPPH radical scavenging assay
The scavenging activity (SA) of AA, 5,6-CA–AA, 2-CA–AA and TBHQ against
DPPH radical were assessed as described in the previous method [15]. Briefly,
DPPH (0.2 mM, 2 mL) was mixed with different concentrations of the tested
compounds (20–100 lM in EtOH, 2 mL) and the reaction was carried out at 25 °C
for 30 min. Then, the absorbance at 517 nm was denoted as A1. Similarly, ethanol
(2 mL) was used to replace the sample as the control. SA was measured according
to the following formula.
ðA0 À A1Þ
SA ð%Þ ¼
Â100%
A0
where A1, A0 are the absorbance of the sample and control, respectively.
123

A. Hui et al.
Hydroxyl radical scavenging assay
The SA of AA, 5,6-CA–AA, 2-CA–AA and TBHQ against hydroxyl-free radicals
were assessed by a modified Fenton reaction protocol [16]. Briefly, 1,10-
phenanthroline (0.75 mM in ethanol, 1 mL) and PBS (pH 7.4, 1 mL) was mixed
with FeSO4 solution (0.75 mM, 1 mL) and the tested compound (2–10 mM in
EtOH, 1 mL), H2O2 solution (0.01%, 1 mL) was added to the above mixture and the
reaction was carried out at 37 °C for 1 h. Deionized water (1 mL) was used to
replace H2O2 as the blank (A00) and 2 mL deionized water was used to replace
H2O2 and the sample as the control (A0). The SA of the tested compound was
evaluated by the following formula:
ðA1 À A0 Þ
0
SA ð%Þ ¼
Â100%
ðA0 À A0 Þ
0
where A1, A0, and A00 are the absorbance of the sample, control and blank,
respectively.
Antilipid-oxidation capacity of ascorbyl cinnamate in peanut oil
In the experiment, the modified Schaal oven method was adapted to accelerate the
oxidation of the peanut oil at 100 1 °C [7]. The above 5,6-CA–AA, 2-CA–AA
and TBHQ were added to the peanut oil (50 g) at a dose of 0.05%, and the sample
without any special compound was made the control. All the samples were placed in
an oven at 100 °C for 2, 4, 6, 8, 10, 12, 14 and 16 h, respectively. Then, their
respective peroxide values (PV) were measured by iodometric determination [17]
and represented by the average of triplicate samples.
Antimicrobial activity screening
Antibacterial activity using disc diffusion method
The inhibitory effect of 5,6-CA–AA, 2-CA–AA, CA and PS on various microor-
ganisms was determined according to the filter paper disc diffusion method [18, 19].
Escherichia coli, Bacillus subtilis and Staphylococcus aureus were activated in
nutrient agar at 37 °C for 24 h, then diluted 108 cfu/mL with sterile physiological
saline and shocked uniformly. Under aseptic operation, 1 mL bacterial suspension
was coated on a plate. Then, 10 mL medium was added to make it uniform on
standby. Next, a 6-mm sterilized paper disc was loaded onto the surface of the plate,
and the control (DMF and sterile saline, denoted as CK1 and CK2, respectively) or
20 lL diluted compounds [40, 20, 10, 5, and 2.5 mg/mL in DMF (5,6-CA–AA,
2-CA–AA and CA) or saline (PS)] were added dropwise on paper disc. Thus, 800,
400, 200, 100,and 50 lg test compounds were absorbed on the disc and the ultimate
plate was incubated at 37 °C for 24 h. Finally, the zone of inhibition (mm) was
measured.
123

Synthesis and evaluation of novel ascorbyl cinnamate as…
Dual inhibition of ascorbyl cinnamate on microbial growth and lipid oxidation
The antimicrobial and antilipid oxidation effect of ascorbyl cinnamate was assayed
by the ‘‘pork-by-soaking’’ method. The test solution was prepared from the mixture
of 5,6-CA–AA and 2-CA–AA (1:1, m/m), CA or PS dissolved in 1% acetic acid
aqueous solution, and the added amount of reagent was 0.05% (m/v). Each 25 g of
pork hind leg was soaked in the tested solution (50 mL) and the control sample was
treated with 1% acetic acid aqueous solution for 10 min. All the treated pork was
drained, sealed with plastic wrap and stored in a refrigerator at 4 °C for a period of
time. Total numbers of colonies [20] and thiobarbituric acid reactive substance
(TBARS) content [21] were analyzed after 0, 3, 5, 7, and 9 days.
Statistical analysis
Analysis of variance (ANOVA) was carried out using SPSS 19.0 software to test for
any significant differences between the groups. Differences between groups at 5%
(P \ 0.05) were considered significant.
Results and discussion
Synthesis of ascorbyl cinnamate
First, the reaction of AA with cinnamyl chloride was performed in CH2Cl2 at RT
and the conversion was very poor, which was ascribed to the poor solubility of AA
in this solvent. Therefore, DMF was introduced and the reaction was clearly
enhanced. The major product was obtained with 65% isolated yield when it was
carried out in DMF-CH2Cl2 (1:3, v/v) and the molar ratio of AA, cinnamyl chloride
and Et3N was 1:2.0:2.0 (Scheme 1). Next, this product was identified as 5,6-CA–
AA, which was different from that of the in lipase catalysis or chemosynthesis in
dimethylacetamide (DMAc)-CH2Cl2 [12, 13]. They both gave 17 as the major
product instead of 5,6-CA–AA. It should be noted that 17 was also detected as a by-
product in the above synthetic procedure [14].
Subsequently, the influence of cinnamyl chloride dose, substrate concentration
and reaction temperature on the preparation of 5,6-CA–AA was investigated.
Unfortunately, these efforts did not bring evident gains. Finally, 5,6-CA–AA was
still the main product of the reaction and 76% yield was achieved in DMF-CH2Cl2
(2:3, v/v).
Similarly, the synthesis procedure of 2-CA–AA (7, Scheme 1) was also studied.
Among the three steps, the second esterified product 20 was gained with better yield
when it was performed in THF/CH2Cl2 and the molar ratio of 5,6-O-isopropylidene-
L-ascorbic acid 19, cinnamyl chloride and Et3N was 1:1.5:1.5. Then, compound 20
was directly de-protected under the optimal pH of 1, resulting in 2-CA–AA with
54% total yield. This result was better than that reported by Galey [6], which gave
only 32.6% overall yield. By comparison, our present synthetic procedure was more
efficient, increasing the output of 2-CA–AA by 65.6%.
123

A. Hui et al.
Stability of ascorbyl cinnamate in solution
The absorbance of AA, 5,6-CA–AA and 2-CA–AA at kmax during storage is
demonstrated in Fig. 4. With the extension of the storage period, the absorbance of
AA decreased from the initial 0.661 to 0.103, which was consistent with that of AA
instability in solution. As for ascorbyl cinnamate, they were both relatively stable.
Thus, a further stability assay was conducted in EtOH-PBS. Within 30 days, almost
no significant changes were observed, their RSD of absorbance being only 1.58 and
0.95%, respectively, as shown in Table 1. The above results indicated that the
stability of 5,6-CA–AA and 2-CA–AA was greatly improved.
In vitro anti-oxidant activity of ascorbyl cinnamate
The DPPH and hydroxyl radical scavenging assay are two of the most common
methods for assessing anti-oxidant activities [22]. The above synthesized com-
pounds were evaluated as anti-oxidant agents in comparing to TBHQ, a perfect anti-
oxidant in lipid system. The anti-oxidant activity of each compound was expressed
in terms of EC50 (the effective molar concentration required to scavenge 50% of
DPPH or hyrdorxyl radical). The SA of AA, 5,6-CA–AA, 2-CA–AA and TBHQ
against DPPH or hydroxyl radical were measured and the corresponding EC50
values are shown in Table 2. In the DPPH radical scavenging test, 5,6-CA–AA
almost maintained the inherent scavenging ability of AA (EC50: 63.96 vs.
59.78 lM) even though ascorbyl cinnamate exhibited poorer capacity than that of
TBHQ (EC50: 46.31 lM). Also, it was noted that the EC50 of AA (59.78 lM,
10.5 lg/mL) under this condition was not entirely in accordance with the data
reported in the literature [23–25], which gave 55, 39.81,and 5.58 lg/mL respec-
tively. The discrepancy was likely because of different concentration ranges,
sampling times and investigators. However, a delightful fact was observed in the
hydroxyl radical scavenging assay in that 5,6-CA–AA was superior to that of TBHQ
Fig. 4 Effect of storage time on the absorbance of AA and ascorbyl cinnamate
123

Synthesis and evaluation of novel ascorbyl cinnamate as…
Table 1 Effect of storage time
Time (d)
5,6-CA–AA
RSD (%)
2-CA–AA
RSD (%)
on the absorbance of ascorbyl
cinnamate
1
0.563
0.571
0.582
0.577
0.558
0.562
0.576
0.483
0.481
0.485
0.479
0.491
0.478
0.487
5
10
15
20
25
30
1.58
0.95
Table 2 EC50 for DPPH and hydroxyl inhibition of chemical compounds
Compound
EC50 for DPPH (lM)
EC50 for hydroxyl radical (mM)
5,6-CA–AA
2-CA–AA
AA
63.96 0.18
81.19 0.21
59.78 0.09
46.31 0.10
7.84 0.05
9.72 0.03
5.78 0.05
8.38 0.09
TBHQ
(EC50: 7.84 vs. 8.38 mM). If a comparison was made between ascorbyl cinnamate
and AA, ascorbyl cinnamate exhibited poorer capacity than that of AA (EC50:
5.78 mM), which was similar to the previous report of ascorbyl fatty acid esters
[26]. In a word, a steady trend was that 5,6-CA–AA showed better anti-oxidant
capacity than that of 2-CA–AA whether in the DPPH or hydroxyl radical
scavenging tesst, which was consistent with that of vitamin C phosphodiesters
linked at the C-2 hydroxyl group with lower anti-oxidant activity [3].
Ascorbyl cinnamate exhibited relative stability in solution and medium anti-
oxidant activity in the above chemical system. Subsequently, it was added to an oil-
soluble system for its antilipid-oxidation evaluation. Peroxide value (PV) is an
indicator to control oily feed, food quality and safety, a lower PV in lipid oxidation
means better anti-oxidant activity of a substrate. Thus, ascorbyl cinnamate, TBHQ,
was added to peanut oil and stored at an elevated 100 °C. As the results show in
Fig. 5, the hydroperoxides formation was remarkably retarded by the compounds
such as 5,6-CA–AA, 2-CA–AA and TBHQ, especially in the duration of 10–16 h
(P \ 0.01, any one vs. control). When the duration reached 16 h, the corresponding
PVs level decreased from an initial 90.13 meq/kg to 61.27, 68.86, and 43.04 meq/
kg, respectively. As seen from the data, TBHQ gave the outstanding anti-oxidation
with 52.2% inhibition. Next, a decrease of 32.0% in PV was obtained in that of 5,6-
CA–AA. On the other hand, there was no significant difference in the anti-oxidant
effect between the two derivatives of AA during the first duration of 8 h (P [ 0.05,
5,6-CA–AA vs. 2-CA–AA). But with the lipid oxidation continued, 5,6-CA–AA
showed stronger anti-oxidant capacity than that of 2-CA–AA (P \ 0.01, 5,6-CA–
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A. Hui et al.
Fig. 5 PVs of peanut oil in the presence of ascorbyl cinnamate at 100 °C
AA vs. 2-CA–AA, maintained for 10–16 h), which was in line with the sequence of
the DPPH and hydroxyl radical scavenging assays above mentioned.
Antimicrobial activity of ascorbyl cinnamate
Since CA has antibacterial activity [9, 10], the inherent property was likely to be
transplanted into these two ascorbyl cinnamate. Therefore, the inhibition of 5,6-CA–
AA and 2-CA–AA against Escherichia coli, Bacillus subtilis and Staphylococcus
aureus were assessed, using CA as a reference and PS as standard antimicrobial
agent. PS, the water-soluble salts of sorbic acid, is an effective and widespread
preservative used in many foods such as meat, drink and cheese [27, 28]. The
antimicrobial results are represented as the zone of inhibition (mm) in Table 3.
Overall, the four test compounds had demonstrated medium to good antimicrobial
activity against both Gram-positive and Gram-negative bacteria and the effect was
dose-dependent. Among them, the antimicrobial ability of PS was the most
prominent no matter what type of microbes. Another obvious trend was that the
effect of 5,6-CA–AA or 2-CA–AA was not less than that of CA when the absorptive
amount ranged from 200 to 800 lg, which showed that the inherent antimicrobial
property of CA was well migrated to the AA derivatives. However, there were some
differences in the performance of the two AA derivatives: 2-CA–AA possessed a
relatively weaker antimicrobial ability than that of 5,6-CA–AA in the assays of
Escherichia coli or Bacillus subtilis, which did not affect its potential application as
an antimicrobial agent since esters had a wide range of active pH over the acids
range [29].
In view of the fact that the two AA derivatives had medium antibacterial activity
against exogenous strains and antilipid oxidation in peanut oil, they may be
potential bifunctional compounds inhibiting microbial growth and fat oxidation.
Based on this consideration, pork was chosen as a suitable system to evaluate the
dual effect of the ascorbyl cinnamate. Meanwhile, PS is also considered as a
123

Synthesis and evaluation of novel ascorbyl cinnamate as…
Table 3 Inhibition zone of 5,6-CA–AA, 2-CA–AA and CA (mm)
Species
Compound
Absorptive amount (lg)
Control
800
400
200
100
50
CK1 CK2
Escherichia coli
5,6-CA–AA 16
13
12 (
9
12
9
8 (
8 (
–
)
)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
2-CA–AA
CA
15
11
24
)
–
8 (
17
16
11(
10
15
8 (
9 (
9 (
17
)
–
PS
19
17
14
11
20
12
12
10
20
13
11
–
8 (
–
)
Bacillus subtilis
5,6-CA–AA 18 ( )
2-CA–AA
CA
16
12
22
)
–
9
–
PS
13
–
10 (
–
)
)
Staphylococcus aureus 5,6-CA–AA 15
)
)
)
2-CA–AA
CA
16
12
23
–
–
8 (
13
)
–
PS
10 (
Antimicrobial ring is fuzzy; – no antimicrobial ring
positive referent compound because it has been listed as a preservative and anti-
oxidant in the ‘‘National Food Safety Standard for Uses of Food Additives’’ (GB
2760-2014) [30]. TBARS content is widely used as an indicator of the degree of
lipid oxidation. In general, people can feel the peculiar smell once the TBARS is
more than 0.5 mg/kg [31]. Therefore, the total number of colonies and TBARS
content of the pork treated with ascorbyl cinnamate, CA, and PS was measured on
different storage days, as shown in Table 4. It was clear that the microbial
reproduction of the pork had slowed down after soaking in any solution of the test
compounds. Among the three compounds, the inhibitory effects induced by ascorbyl
cinnamate or PS were significant during the storage (P \ 0.01, mixed ascorbyl
cinnamate vs. control; P \ 0.01, PS vs. control). In contrast, the inhibition of CA
was relatively obvious only in the storage of 5–9 days (P \ 0.01, P \ 0.05, CA vs.
control). A similar phenomenon was found in the result of TBARS content. The use
of any special compound greatly retarded the TBARS level of the pork [P \ 0.01,
ascorbyl cinnamate (PS) vs. control; P \ 0.05, P \ 0.01, CA vs. Control].
Moreover, the sample treated with mixed ascorbyl cinnamate had a lower TBARS
content than that of PS during the storage, but the difference was significant only on
the fifth or ninth day (0.20 0.03 vs. 0.28 0.01, P \ 0.05; 0.45 0.02 vs.
0.54 0.06, P \ 0.05). Taking the TBARS level of the fifth day as an example, a
decline of up to 56.5% was found in the pork treated with ascorbyl cinnamate.
Under the same conditions, the application of PS only gave a 39.1% reduction
compared with control.These results indicate that ascorbyl cinnamate exhibited a
promising dual inhibition on the lipid oxidation and microbial reproduction
although its preservative effect on pork was slightly weaker than that of the positive
referent compound (P \ 0.05, ascorbyl cinnamate vs. PS, on the fifth, seventh, and
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A. Hui et al.
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Synthesis and evaluation of novel ascorbyl cinnamate as…
ninth days). Also, the derivative modified in the hydroxyl position of AA such as
5,6-CA–AA may be a potential bifunctional candidate in future food, cosmetics or
drug trials.
Conclusion
Two novel ascorbyl cinnamates, 5,6-CA–AA and 2-CA–AA, were successfully
prepared with 76 and 54% yields, respectively. Of the two, 5,6-CA–AA was first
synthesized and an efficient access to 2-CA–AA was developed with a 65.6%
increase in the yield. The subsequent investigation indicated that 5,6-CA–AA
maintained the inherent anti-oxidant function of AA. Also, 5,6-CA–AA and 2-CA–
AA possessed antimicrobial activity as well as CA in vitro. The application of
ascorbyl cinnamate could effectively retard the lipid oxidation of peanut oil or pork,
and the microbial reproduction of pork was inhibited to some extent. This work
offers an excellent approach for the derivation of AA, which may lead to the
discovery of potent efficient bifunctional compounds.
Acknowledgements The authors are grateful to National Natural Science Foundation of China (Nos.
21302037 and 81303245) and Anhui Science and Technology Major Project (No. 16030701072) for
financial assistance.
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