Evaluation of Chinese Prickly Ash and Cinnamon to Mitigate Heterocyclic Aromatic Amines in Superheated Steam-Light Wave Roasted Lamb Meat Patties Using QuEChERS Method Coupled with UPLC-MS/MS

: Chinese prickly ash and cinnamon contain many antioxidants, which scavenge free radicals and can reduce many harmful compounds, such as heterocyclic aromatic amines (HAAs). Modern technologies used for cooking, such as the use of superheated steam roasting, are beneﬁcial in decreasing the development of HAAs. The current study was based on the use of these two spices in roasted lamb patties to mitigate the formation of HAAs in superheated steam roasted patties. Results exhibited signiﬁcant differences ( p < 0.05) in the content of both polar and non-polar HAAs as compared to control patties. In cinnamon roasted patties, polar HAAs were reduced from 23.76 to 10.56 ng g − 1 , and non-polar HAAs were reduced from 21.34 to 15.47 ng g − 1 . In Chinese prickly ash patties, polar and non-polar HAAs were 43.60 ng g − 1 and 35.74 ng g − 1 , respectively. Similarly, cinnamon-treated patties showed a signiﬁcantly higher ( p < 0.05) reduction in polar HAAs (23.52 to 12.41 ng g − 1 ) than non-polar (16.08 to 9.51 ng g − 1 ) at concentrations of 0.5–1.5%, respectively, as compared to the control, with 45.81 ng g − 1 polar and 35.09 ng g − 1 non-polar HAAs. The polar HAAs tested were PhIP, DMIP, IQx, and 8-MeIQx, while the non-polar were harman and norharman. Both spices and superheated steam controlled HAAs to a signiﬁcant level in lamb meat patties.


Introduction
Heterocyclic aromatic amine (HAA) formation depends on heat transfer, lipid degradation, oxidation, and cooking methods, such as barbecuing and grilling [1]. Heterocyclic amines can be reduced by the application or induction of antioxidants, as well as the modification of cooking methods [2]. The addition of antioxidants to meat has been considered to be an effective strategy to reduce HAA exposure because of the hypothetical free radical pathway leading to HAA formation [3].
According to Adeyeye [4], antioxidants may trap free radicals, such as intermediates of HAAs, to prevent the formation of HAAs. Among spices, cinnamon and Chinese prickly ash are good sources of antioxidants in meat and meat products in Asian countries [5]. In a lipid peroxidation assay test by Thaipong [6], it was estimated that cinnamon showed more significant activity than anise, ginger, licorice, nutmeg, or vanilla. Chinese prickly ash is also consumed in Central Asian countries, such as China, and is used as an important spice [7]. Antioxidant compounds, such as sanshools and sanshoamides, are very beneficial compounds in this spice, and are responsible for its antioxidant activity [8]. The spices can be a beneficial strategy to control HAAs in cooked meat products [9].
Modern cooking methods can be more helpful in the reduction of HAAs than conventional barbecuing or grilling, which include direct contact of the meat with flame [10]. Modern cooking methods, such as infrared grilling and superheated steam-light wave roasting, offer great potential to reduce HAAs in cooked lamb meat products [11]. One new technology introduced superheated steam-light wave roasting, which is applied by using water vapor to form steam in the oven, and has a higher temperature, which can be helpful in controlling HAAs at a very significant level [12]. The study aimed to analyze the effect of spices and superheated steam-light wave roasting to inhibit HAAs in roasted lamb meat patties.

Lamb Meat and Spices
A total of 12 fresh lamb shoulder oyster cut muscles of 8 month old sheep were obtained from the Hongbao sheep meat industry of Bayannur, Inner Mongolia, China. The spices, cinnamon powder and Chinese prickly ash powder, were bought from the local spice market in Beijing, China [5].

Determination of DPPH Activity of Spices
The antioxidant activity of spices was determined using the DPPH scavenging activity of spices, following the method of Thaipong [13]. Three different concentrations (10, 20, and 30 uL/mL) of Chinese prickly ash and cinnamon were prepared. From each concentration of spices, 1 mL of extract solution was added to 2 mL of freshly prepared DPPH solution (0.1 mM in 95% methanol). This mixture was vortexed for 10 s and then placed in a dark place for 30 min. The absorbance was measured at 517 nm wavelength using the UV-vis spectrophotometer (Persee TU-1810 UV-vis; Persee Instruments Co., Ltd., Beijing, China) at room temperature. Lower absorbance indicated higher radical-scavenging activity. Radicalscavenging activity was calculated as the percentage of DPPH discoloration using the following equation: DPPH-radical-scavenging activity % = 100 where AE represents the solution absorbance at 517 nm when 1 mL of each spice solution was mixed with 2 mL of 0.1 mmol·L −1 DPPH solution after incubation (30 min) at room temperature, and AD represents the absorbance of 2 mL of 0.1 mmol·L −1 DPPH solution with 1 mL Milli-Q H 2 O. The final unit is % DPPH activity of the sample that has decreased the DPPH content by scavenging the radicals.

Preparation of Lamb Patties with Spices
After the sheep were slaughtered, their carcasses were cooled at 4 • C for 24 h. Later, the shoulder oyster muscles were removed from the carcasses and kept at −80 • C until they were employed in further experiments. To make the patties, slices of the meat from the lamb oyster muscles were used. It was ensured that visible fat from muscles was properly removed. The 12 muscles were individually ground using a grinder with 5 mm blades to produce ground meat and ensure proper grinding of each muscle. Each muscle weighed 10 g after being ground. Fifty grams of fresh ground lamb meat was taken, and spices (cinnamon and Chinese prickly ash) were added in concentrations of 0.5%, 1%, and 1.5% to the ground meat paste to create each patty, using a 6 cm × 1.5 cm mold to make patties of the same size. The patties with two spices were separately prepared in triplicates. Three patty treatments contained spices, and one without spice was used as control. There were 12 patties in each spice-treated group; therefor, a total of 24 patties were made for each spice (cinnamon and Chinese prickly ash) [5].

Cooking of Patties
The spiced lamb patties were cooked using a superheated steam-light wave roasting method at 240 • C. The patties were roasted for 17 min, which was needed to reach a core temperature of 72 • C in the oven. The patties were cooked in two batches; one batch for Chinese prickly ash and the other for cinnamon. A digital data logger (Hangzhou Co., Ltd., Hangzhou, China) with a digital thermometer probe was used to observe the internal temperature of each patty. The lamb patties were further cooled at room temperature for one to two hours, and then packed in zip-lock bags and stored at −20 • C until used [11].

Determination of HAAs in Roasted Patties
HAAs in roasted patties were determined using the method developed by Hsiao, Chen, and Kao [14]. Two grams of ground patty sample were obtained and places in a centrifuge tube. A total of 10 mL of deionized water and one ceramic stone were placed inside the centrifuge tube containing the sample. The tubes were then mixed for 10 min. After adding 10 mL of acetonitrile containing 1% acetic acid, the tube was shaken once more for 10 min. After mixing, 4 g of anhydrous MgSO 4 and 1 g of anhydrous C 2 H 3 NaO 2 were dispersed. After 1 min, the centrifuge tube was spun at a speed of 3200× g of relative centrifugal force for 10 min at a temperature of 4 • C (RCF). A tube containing 900 mg of anhydrous MgSO 4 , 300 mg of propylsulfonic acid modified silica (PSA), and 300 mg of C18 was used to purify the supernatant after 10 min of centrifugation. The tube was oscillated for 1 min and centrifuged at 3200× g, at 4 • C, for 5 min.
One milliliter of the supernatant was removed after 1 min of centrifugation and nitrogen was used to freeze-dry it. The freeze-dried sample received 0.2 mL of methanol, which was then vortexed. The material was then filtered using a polyvinylidene difluoride (PVDF) membrane filter with a pore size of 0.22 µm. The samples were then analyzed using UPLC-MS/MS (Agilent model 1290). Mass spectrometric analyses were performed on an AB Sciex API 4000™ triple quadrupole mass spectrometer equipped with an electrospray ionization source to look for heterocyclic aromatic amines [15]. Separation was achieved on a Shim-pack GIST C18 (2.1 × 100 mm, 3 µm, 100 Å) at 37 • C. For further dilution, standard stock solutions containing 10 mg in 5 mL methanol were prepared. To establish calibration curves, LODs, and LOQs, stock solutions of standards mixed solutions with final concentrations of 5, 10, 25, 50, 100, 300, and 500 ppb in methanol were created. The mobile phase for analysis at UPLC-MS/MS was composed of (A) 100% HPLC/UPLC-grade acetonitrile and (B) 10 mM ammonium acetate solution (pH 2.9) [16]. For the purpose of achieving equilibrium in the column, a linear gradient profile with 85% A and 15% B was maintained for the first 8 min, changing to 45% A and 55% B after 13 min, and then to 91% A and 9% B after 16 min. For the analysis, a column temperature of 25 • C was maintained with a flow rate of 0.4 mL/min. The injection volume for each sample's analysis was 2 µL. By using UPLC-MS/MS, each sample was examined for 30 min. Table 1 shows the limits of detection and limits of quantification for all HAAs compounds detected through UPLC-MS/MS. The linear range of 0.1-5.00 ng g −1 and recovery was from 54.86% to 108.32% for all HAA standards. Figure 1 shows the peaks of the standards obtained by UPLC-MS/MS. The internal standard was a mixture of 20 HAA standards, and was used for the analysis of HAAs.  Figure 2 shows the DPPH activity of cinnamon and Chinese prickly ash. Both spices showed significant antioxidant activity. Cinnamon showed inhibition rates of 91.96%, 92%, and 91.66% at concentrations of 10, 20, and 30 µL/mL, respectively, while Chinese  Figure 2 shows the DPPH activity of cinnamon and Chinese prickly ash. Both spices showed significant antioxidant activity. Cinnamon showed inhibition rates of 91.96%, 92%, and 91.66% at concentrations of 10, 20, and 30 µL/mL, respectively, while Chinese prickly ash showed inhibition rates of 86.23%, 87.96%, and 89.4% at concentrations of 10, 20, and 30 µL/mL, respectively, which was lower than cinnamon.  Figure 2 shows the DPPH activity of cinnamon and Chinese prickly ash. Both spices showed significant antioxidant activity. Cinnamon showed inhibition rates of 91.96%, 92%, and 91.66% at concentrations of 10, 20, and 30 µL/mL, respectively, while Chinese prickly ash showed inhibition rates of 86.23%, 87.96%, and 89.4% at concentrations of 10, 20, and 30 µL/mL, respectively, which was lower than cinnamon.  Table 2 lists the findings of the polar HAAs found in the patties treated with Chinese prickly ash. Due to the high antioxidant capacity of Chinese prickly ash, it was shown that its inclusion reduced the production of several polar HAAs [16]. Some polar HAAs, such IQ (4,5-b) and 7,8-DiMeIQx, were not found in detectable amounts. Other polar HAAs,  Table 2 lists the findings of the polar HAAs found in the patties treated with Chinese prickly ash. Due to the high antioxidant capacity of Chinese prickly ash, it was shown that its inclusion reduced the production of several polar HAAs [16]. Some polar HAAs, such IQ (4,5-b) and 7,8-DiMeIQx, were not found in detectable amounts. Other polar HAAs, such as IQx, IQ, and 4,8-DiMeIQx, were found in low concentrations; their respective concentrations ranged from 3.25 to 1.91 ng g −1 , 0.16 to 0.12 ng g −1 (at 1.5% there was no detection of IQ), and 0.78 to 0.38 ng g −1 . Results are given as means ± standard errors with superscripts (a, b, c) in columns showing significant differences (p < 0.05) within treatments of lamb patties. In beef patties which had Chinese prickly ash also showed a reduction in the polar and the non-polar HAAs [17].

Quantity of Polar and Non-Polar HAAs in Superheated Steam-Light Wave Roasted Patties and Chinese Prickly Ash
According to the Table 3 findings, the level of non-polar HAAs, such as harman and norharman, which ranged from 8.89 to 6.66 ng g −1 and 9.28 to 6.08 ng g −1 , respectively, was greater in lamb patties [18]. The concentration of Glu-P-1 ranged from 2.10 to 1.66 ng g −1 , and that of Glu-P-2 from 1.29 to 1.12 ng g −1 . The lowering of both types of HAA content was observed to be positively impacted by Chinese prickly ash [19]. The Chinese prickly ash-treated patties had lower levels of HAAs than the control samples. According to Figure 3, control patties contained 35.74 ng g −1 of non-polar HAAs and 43.60 ng g −1 of polar HAAs.   Results are given as means ± standard errors with superscripts ( a , b , c ) in columns showing significant differences (p < 0.05) within treatments of lamb patties.
that of Glu-P-2 from 1.29 to 1.12 ng g . The lowering of both types of HAA content was served to be positively impacted by Chinese prickly ash [19]. The Chinese prickly ash-tre patties had lower levels of HAAs than the control samples. According to Figure 3, control ties contained 35.74 ng g −1 of non-polar HAAs and 43.60 ng g −1 of polar HAAs. Results are given as means ± standard errors with superscripts ( a , b , c ) in columns showing signifi differences (p < 0.05) within treatments of lamb patties.  In comparison to the control, the Chinese prickly ash-treated patties had lower concentrations of polar (23.76 ng g −1 ) and non-polar (21.34 ng g − 1) HAAs at 0.5%. At 1%, polar HAA concentrations were marginally lower (20.69 ng g −1 ), while non-polar HAA concentrations were found to be 17.73 ng g −1 . Chinese prickly ash patties contained 10.56 ng g −1 of polar HAAs at a concentration of 1.5%, while 15.47 ng g −1 of non-polar HAAs were present. The findings demonstrated that the content of the polar HAAs varied significantly between concentrations, as well as when compared to control patties. With the exception of 1.5% concentration, where total non-polar HAAs decreased relative to total polar HAAs, the overall content of polar HAAs was somewhat higher than non-polar HAAs [5]. Table 4 displays the results of the polar HAAs in lamb patties with cinnamon added in various amounts. The polar HAAs were found and contrasted with the no-spice control. The outcomes revealed that all polar HAA levels were greater in the control group [20]. Although there was a significant difference (p < 0.05) among the contents seen in the results, they were consistent with the other two spices studied in [21]. The highest values among polar HAAs were observed in 8-MeIQx, DMIP, and PhIP, which ranged from 11.80 to 4.05 ng g −1 , 2.93 to 2.24 ng g −1 , and 4.47 to 3.16 ng g −1 , respectively, and corresponded to treatments 0.5%, 1%, and 1.5% cinnamon powder, respectively. IQ (4,5-b), IQ, ISO-IQ, and 7,8 DiMeIQx content was too low at 1% and 1.5% to be recognized by suppression of cinnamon antioxidants in comparison to the control, while the content was still visible in roasted patties [22]. IQx and 4, 8-DiMeIQx levels were also very low among polar HAAs in the patties with the addition of cinnamon powder, with content reduced from 2.91 to 2.26 ng g −1 and 0.98 to 0.68 ng g −1 , or from 0.5% to 1.5% concentration, in cinnamon patties. The results of non-polar HAAs, reported in Table 5 below, show that harman and norharman were higher in content than Glu-P-1 and Glu-P-2; harman and norharman levels were 7.65-3.56 ng g −1 and 5.95-3.89 ng g −1 , respectively, from 0.5% to 1.5%, while the content of Glu-P-1 and Glu-P-2 was detected to be 1.37-1.03 ng g −1 and 1.28-1.02 ng g −1 , respectively. The results showed that the amount of the polar HAAs was higher generally, and, individually, harman and norharman showed higher content at each concentration, but there was a reduction at all concentrations as compared to content observed in the control. Table 5. Non-polar HAAs (ng/g) in the cinnamon powder-treated patties with superheated steamlight wave roasting.

Treatments
Glu-P-1 Norharman Harman Glu-P-2 Cinnamon powder has shown a very potent activity towards the reduction of HAAs after addition in different concentrations [23]. At all concentrations, cinnamon powder had a diminishing effect on the content of HAAs, while the control patties, without cinnamon, had higher contents; the content of polar HAAs was 45.81 ng g −1 , while the non-polar HAA content was 35.09 ng g −1 , as shown in Figure 4. At 0.5% concentration of cinnamon powder added to the lamb patties, the content of polar HAAs was 23.52 ng g −1 , while for non-polar HAAs content was 16.08 ng g −1 . At 1%, the content of the polar HAAs was 19.78 ng g −1 , while for non-polar HAAs it was 14.04 ng g −1 . At 1.5% concentration of cinnamon powder, the content of polar HAAs was 12.41 ng g −1, while for non-polar HAAs it was 9.51 ng g −1 .
Overall, the results showed that polar HAAs were higher in content both in control as well as in the treated patties with cinnamon powder. Cinnamon powder has shown a very potent activity towards the reduction of HAAs after addition in different concentrations [23]. At all concentrations, cinnamon powder had a diminishing effect on the content of HAAs, while the control patties, without cinnamon, had higher contents; the content of polar HAAs was 45.81 ng g −1 , while the non-polar HAA content was 35.09 ng g −1 , as shown in Figure 4. At 0.5% concentration of cinnamon powder added to the lamb patties, the content of polar HAAs was 23.52 ng g −1 , while for non-polar HAAs content was 16.08 ng g −1 . At 1%, the content of the polar HAAs was 19.78 ng g −1 , while for non-polar HAAs it was 14.04 ng g −1 . At 1.5% concentration of cinnamon powder, the content of polar HAAs was 12.41 ng g −1, while for non-polar HAAs it was 9.51 ng g −1 . Overall, the results showed that polar HAAs were higher in content both in control as well as in the treated patties with cinnamon powder.

DPPH Activity of Cinnamon and Chinese Prickly Ash
Antioxidant compounds present in foodstuffs play a vital role in human life, acting

DPPH Activity of Cinnamon and Chinese Prickly Ash
Antioxidant compounds present in foodstuffs play a vital role in human life, acting as health-protecting agents. In addition to this role, antioxidants are one of the key additives used in fats and oils [24]. Cinnamon is a very popular spice used in many foods as a flavor additive. It has many other beneficial properties as well, such as anti-inflammatory and antioxidant characteristics [25]. China uses various traditional spices for aroma and taste. One of the most popular spices is Chinese prickly ash, which is added to meat dishes for taste and aroma [7,26]. Based on our results, cinnamon and Chinese prickly ash are both good sources of natural antioxidants, as both showed good or higher levels of inhabitation of DPPH; levels were almost as high as BHT, which is an artificial antioxidant source. Processed meat is cooked at high temperatures, due to which some harmful compounds are produced, such as d-heterocyclic aromatic amines (HAAs) which can cause cancer. The use of cinnamon and Chinese prickly ash in meat products can prevent and block the oxidation of HAAs because of their high antioxidant activity [5].

Effect of Chinese Prickly Ash Powder and Superheated Steam-Light Wave Roasting on HAAs in Roasted Patties
In China, various traditional spices have become popular around the globe which provide particular taste and aroma in food. Most meat products cooked with these spices are symbolic of Chinese cuisine around the world. One of the most popular spices is Chinese prickly ash (Zanthoxylum bungeanum), which is added to meat dishes for taste and aroma [7,26]. Moreover, it is considered to have many important antioxidants and exhibit anti-inflammatory and anti-cancer activity. It contains mainly sanshools and sanshoamides as the main antioxidant compounds [27]. We found that, due to this property, Chinese prickly ash can very efficiently reduce HAAs in roasted lamb meat. The results of the present study are consistent with another study [18], which showed that Chinese prickly ash decreased the content of all polar HAAs. At concentrations of 0.5% to 1% of Chinese prickly ash in roasted beef patties, the contents of PhIP, DMIP, MeIQx, and 4,8-DiMeIQx decreased from 11 to 6.06 ng g −1 , 0.42 to 0 ng g −1 , 0.69 to 0.32 ng g −1 , and 0.25 to 0.24 ng g −1 , while in non-polar HAAs, harman content was seen to increase from 0.73 ng g −1 to 0.96 ng g −1 , and norharman content from 5.12 ng g −1 to 6.51 ng g −1 , which was opposite to the trend of our results, where we observed a decline of non-polar HAAs at each concentration.
In another study [10], the role of this spice was observed to be very effective in the inhibition of many HAAs in grilled beef. The content of polar HAAs, such as PhIP, was reduced from 1.28 to 0.80 ng g −1 by the addition of 0.5-1.5% concentration of Chinese prickly ash, while IQx was only found at 0.5%, with 0.12 ng g −1 . MeIQx was reduced from 1.13 to 0.33 ng g −1 with a 0.5% to 1.5% concentration of Chinese prickly ash. The content of 4,8-DiMeIQx was reduced from 0.04 ng g −1 to 0.02 ng g −1 at concentrations of 0.5% to 1.5%. We detected that content of PhIP, IQx, IQ, and 4,8-DiMeIQx was slightly higher than in this study, but the decrease we observed was similar to that observed in this previous study. For non-polar HAAs, the content of harman was reduced from 2.94 to 2.41 ng g −1 , while norharman was slightly increased from 16.91 to 17.96 ng g −1 at three concentrations, which is quite a contradictory trend compared to our results. In our study, the non-polar HAAs harman and norharman decreased in lamb patties with increasing concentrations of Chinese prickly ash. These results are comparable with those reported by other authors who studied the presence of antioxidant compounds in lamb meat [28].

Effect of Cinnamon Powder and Superheated Steam-Light Wave Roasting on HAAs in Roasted Patties
Cinnamon (Cinnamomum cassia or Cinnamonum zeylanicum), which is a popular spice in South Asian and Central Asian countries, is an ordinary spice used on meat products as a flavoring and aromatic agent [29]. Cinnamon has many other beneficial properties, such as anti-inflammatory and antioxidant characteristics [30]. The beneficial bioactive compounds (cinnamaldehyde, cinnamic acid, cinnamyl alcohol, coumarin, and eugenol) in cinnamon make it an important plant spice for medicinal purposes [31]. It is also used to treat many illnesses, such as cancer and diabetes [32]. [33] stated that cinnamon has the potential to inhibit HAAs in beef. Our findings are consistent with a prior study, which found that cinnamon decreased PhIP in descending order, starting with 0.58 ng g −1 in beef [33]. There are few publications on DMIP in the literature. Here, we discovered that cinnamon powder in lamb meat considerably (p < 0.05) decreased DMIP. In another study [34], it was shown that the addition of cinnamon, at 0.5% concentration, to cooked beef reduced the content of IQ to a lower content than all tested spices. The content of IQ was reduced to the very low level of 0.85 ng g −1 , which is very similar to our results.
Similar results were observed in all concentrations, and the contents were reduced to a significant level in lamb meat by the addition of cinnamon. However, in this study PhIP content was increased to 0.43-1.94 ng g −1 which is surprising, because we observed a significant decline in our results of PhIP and all polar HAAs. However, the contents of MeIQx and IQx were lower than our values of HAAs at 0.5% cinnamon powder. The MeIQx values were consistent with our results, but there was variation in samples, including kind of meat, cooking method, and temperature variation, as well as cooking method used, between the studies.

Conclusions
The spices Chinese prickly ash and cinnamon have great potential as antioxidants, and this property of these spices has an impact on HAA formation. Cinnamon, especially, has great potential to inhibit HAAs, as the antioxidant compounds in it hinders the formation of HAAs. Both kinds of HAAs were reduced by the addition of these spices. Moreover, superheated steam-light wave roasting proved to be a cooking method that was very beneficial in the reduction of HAAs at a significant level as compared to other traditional methods, observed in our previous studies. Therefore, we conclude that the use of cinnamon in meat products can be an important natural source of antioxidants; furthermore, the use of superheated steam-light wave roasting can be a beneficial technology to reduce harmful compounds, which needs further exploration in this area of meat science.