Quality analysis combined with mass spectrometry imaging reveal the difference between wild and cultivated Phyllanthus emblica Linn.: From chemical composition to molecular mechanism

2.1 Ethics statement

Volunteers were given written informed consent forms about the purpose of the study and their right to keep information confidential. Informed written consent was obtained from all participants.

2.2 Materials and chemicals

Water was purified using a Milli Q water purification system (Millipore, Bedford, MA, USA). HPLC-grade methanol was purchased from Fisher Chemical (Pittsburg, PA, USA). HPLC-grade formic acid was obtained from Chengdu KeLong Chemical Factory (Chengdu, China). Anhydrous Ethanol (Analytical purity), Vitamin C was also purchased from Chengdu KeLong Chemical Factory (Chengdu, China). DPPH free radical scavenging ability test kit, ABTS buffer solution was purchased from Solaribio biotechnology Co., Ltd. (Beijing), α-Glucosidase, 4-Nitrophenyl-β-D-glucopyranoside (PNPG, Sigma, USA) and Acarbose (Bayer, Germany). Standards of Citric acid, mucinous acid, malic acid Gallic acid (GA, No. CHB201131), Epicatechin gallate (ECG, No. CHB-B-081), Quercetin(Q, No. CHB-H-040), Corilagin (CR, No. CHB-K-004), Gallocatechin (GC, No.4051109), Catechin (C, No.14051508), Epigallocatechin gallate (EGCG, No.14121608), Gallocatechin gallate (GCG, No.14102009), Ellagic acid (EA, No. CHB-R-039), Chebulagic acid (CLA No. CHB-H-114), Chebulic acid (CA No. CHB-H-140), Chebulinic acid (CBA No. CHB-H-018) were purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). The purity of the twelve standards was each above 98.0%.

2.3 Phyllanthus emblica sample collection

12 batches of PE samples were collected from Yunnan and Guangdong, and identified by associate professor Gao Ji-hai from Chengdu University of TCM, and the samples are kept in the National Seed Resource Bank. The specific sampling information is shown in Table 1 and Fig. 1. All the 12 batches were fresh PE fruits collected at mature period.

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Fig. 1

Typical figures of PE plant (A), appearance of wild and cultivated PE (B).

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2.4 UPLC-QTOF-Mass conditions

2.5 Mass imaging conditions

PE was cut into even sheet and frozen In −80  degreesC refrigerator. After removal, the slide was covered and compressed, and then freeze-dried as the mass spectrum imaging sample. Samples were analyzed by SYNAPT XS, with DESI ion source. Mass spectrometry conditions were set as follows: negative ion scanning, scanning range100-1500 m/z, spatial resolution 120 um, spray voltage 4.5KV; sampling cone voltage 40 V; sprayer (nitrogen pressure) 0.5 MPa; solvent methyl formic acid (1000: 1), volume flow 2 μL/min; sprayer angle of incidence 70 degrees; collector angle 10 degrees; ion source temperature 150℃; sampling voltage was −40 V. The data acquisition software was Masslynx V4.2 (Waters company, USA) and data processing software was Masslynx HDI V 1.5 software (waters company, USA).

2.6 Establishment of temporal dominant description method

To evaluate the taste difference of PE, a human sensory test using the visual analog scale (VAS) was proposed to verify the results (Han et al., 2018)(Han, Jiang, Han, Xiong, He, Fu, et al., 2018). VAS was a measurement instrument for subjective characteristics or attitudes that cannot be directly measured. It was initially widely used in pain scoring, and then used in the sensory evaluation. With the approval of the medical ethics committee of the Affiliated Hospital of Chengdu University of TCM, 10 well-trained and healthy volunteers (4 males and 6 females, aged 21–28) were selected. Volunteers were selected from graduate students at Chengdu University of TCM. They have no smoking, drinking and drug abuse, no genetic history, no recent oral and throat diseases, and normal taste. All the volunteers were voluntary and signed the informed consent before the experiment.

PE has four basic flavors, which are astringency, bitterness, sourness, aftertaste-sweetness, and one sensations called saliva secretion. Among them, aftertaste-sweetness and salivary secretion are important sensory characteristics of PE. These flavors and sense will appear in sequence during the chewing process, and the last one is sweetness, which is called aftertaste-sweetness. Its precise meaning is that in a certain period of time, a certain taste will occupy an absolute advantage, which makes the flavor of PE have a sense of hierarchy. Previous sensory descriptions cannot accurately evaluate the temporally hierarchical taste of PE. Therefore, it is necessary to establish a special method for PE taste and flavor evaluation, which is called temporal dominant description method (P. Li, Zhang, Lin, Han, Ke, Han, et al., 2019). During the training sessions, volunteers were trained with different concentrations of model solutions (Table 2) so they were accustomed to the evaluation scales and bitterness intensities. After that, the samples were evaluated. A drop of approximately 10 mL of each solution was applied to the upper surface of the tongue for 15 s. Then, the test solution was expectorated. Volunteers were asked to score the “bitterness, sweetness, astringency, acidity” using the 100 mm VAS by placing a mark along a 100 mm line 23. Between each test interval, the mouth was rinsed well with distilled water so that no bitter taste remained. Volunteers were given a break between each session.

2.7 HS-SPME/GC-QQQ-MS/MS conditions

2.8 Biological activity determination methods

Take the α-glucosidase and PNPG reaction system as a model for testing, and the specific operations were as follows: Added 10 μL of α-glucosidase solution (2U/mL) and 10 μL of the sample solution to each reaction well in turn, mixed well and incubated in a 37 ℃ water bath for 15 min. Then, added 50 μL of PNPG (1 mmol/L), placed it in a 37 ℃ water bath and incubated for 30 min. Finally, added sodium carbonate solution to stop the reaction. Each sample has 3 replicate wells. Measured the absorbance at 405 nm as soon as possible by a multifunctional 96-well plates reader, and repeat for 3 times. Acarbose was used as a positive control, the concentration was 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 mg/mL; the samples were set with 8 concentration gradients of 50, 100, 150, 250, 350, 500, 750, and 1000 μg/mL and calculate the inhibition rate.

According to the DPPH free radical scavenging ability test kit (Solebo biotechnology Co., Ltd.) instructions, prepared the solution and required reagents. Vitamin C was used as a positive control, the concentration was 0.01, 0.05, 0.20, 0.40, 0.60, 0.80, 1.00, 2.00 mg/mL; the samples were set with 8 concentration gradients of 0.20, 0.60, 1.00, 2.00, 4.00, 6.00, 8.00, and 10.00 mg/mL and calculated the scavenging rate. The calculation formula was as follows: $$\mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}(\%)=\frac{A0-(A1-A2)}{A0}\times 100\%$$

In the formula: A₀ was the absorbance value of blank group; A₁ was the absorbance value of inhibition group; A₂ was the absorbance value of background group. All data were imported into Graphpad prism7.0 software to calculate half inhibitory concentration (IC₅₀).

The antibacterial activity of PE is one of its important biological activities. It has a wide range of antibacterial activities, especially against oral bacteria and common pathogenic bacteria(Staphylococcus aureus, Candida albicans and Aspergillus flavus) (Khan, Hassan, Ullah, Karim, Baseer, Abid, et al., 2013; Thaweboon & Thaweboon, 2011). Staphylococcus aureus, Candida albicans and Aspergillus flavus (purchased from Baina Biological Co., Ltd., China) were inoculated and cultured for 3 generations. Under aseptic conditions, take 0.2 mL of bacterial suspension (the best concentration of bacterial solution is 1 × 10⁶ CFU/mL) and spread it evenly on the surface of the agar plate. The positive group was gentamicin sulfate injection (diluted four times). Take 2 mL of each sample solution (12 times water extract, filter sterilization) into a sterilized EP tube, and then put a 6 mm diameter neutral filter paper into the EP tube to soak for 4 h. Place the filter paper clockwise on the same plate, parallel three groups, and measure the average value after incubation at 37 ℃ for 24 h.

2.9 Data processing and analysis

Statistical analyses were performed using SPSS 22.0 package (SPSS Inc., Chicago, IL, USA) and Oringin 2018 (OriginLab, Hampton, Massachusetts, USA). PCA and OPLS-DA were analyzed by SIMCA-P11.0 (umetrics AB, Umea, Sweden).

3.1 Temporal dominant description evaluation results

PE has 5 basic taste senses, which are bitterness, sourness, astringency, aftertaste-sweetness and stimulate saliva secretion. These five sensations represent the five main kinds of compounds, such as the sourness and saliva secretion comes from organic acids, astringency comes from tannins, aftertaste-sweetness is more complex may come from the comprehensive effect of sugars, amino acids and catechins (Das, Sasmal, & Arora, 2021). Meanwhile, these five sensations will appear in sequence at different times after PE enters the oral cavity. Based on the PE taste characteristics, this paper established a temporal dominant description methods to evaluate the sensory characteristics of wild and cultivated PE(P. Li, et al., 2019). First, 10 volunteers’ VAS scores were used to evaluate 5 sensory scores at different time points. The average scores of 12 batches at different time points were shown by heat map, and the results are shown in Fig. 2 A. It can be clearly seen from the results of the heat map that the five senses of taste are obviously time-dependent. In 0–20 s, sourness, bitterness and astringency were the dominant taste senses, while in 20–40 s were aftertaste and saliva secretion. The tastes appearance time is defined as the taste intensity retention time (TIRT). During the whole tasting process, the bitterness, astringency and sourness intensity (I) of PE showed a downward trend, the rest showed a trend of rising first and then falling. It can be intuitively found from the heat map (Fig. 2 A) that the intensity and duration of bitterness, sourness and astringency of wild PE are significantly stronger than that of cultivated PE. However, many evaluation methods like single oral taste evaluation method, fuzzy mathematics comprehensive evaluation method, sensory simulation evaluation method, etc. cannot accurately reflect the taste dynamic changes in the mouth during chewing. Therefore, it is necessary to establish a method to describe its TIRT.

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Fig. 2

Temporal dominant description evaluation analysis and odor component analysis results, (A) the heatmap of average abundance for the volunteer’s VAS scores at 8 time points, (B) PCA result of comprehensive taste index, (C) loading scatter plot of PCA, (D) clustering heat map of T value, (E) PAC analysis result, (F) comparison result of odor intensity mean value of each component.

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It is found through fitting calculation that the sensory intensity and retention time are linearly fitted (y = kx + b), and the fitting results are shown in Table 3. Thus, the slope k value can be easily obtained. The meaning of k value is that the greater the absolute value of k, the shorter the taste retention time. Therefore, it is necessary to establish a function to characterize its retention time. Since the highest taste intensity is 10, the maximum value of the slope (k) is also 10 (the first time point is 0, the second time point is 10, so 0 < │k│〈1 0). There are eight time points in the experiment, and the maximum value is 8. Using the above data, the conversion function f (x) = -0.8x + 8 can be obtained. Here, we introduce its retention time coefficient K to characterize the taste retention time, the formula is as follows: $$K=f\left(x\right)=-0.8\left|k\right|+8$$

K represents the TIRT coefficient of 5 tastes, which is defined as: bitterness (K_B), astringency (K_A), sourness (K_S), aftertaste-sweetness (K_AS) and stimulate saliva secretion (K_SS). In the fitting process, the data starting from 5 s until the taste intensity is greater than or equal to 0.5 are regarded as fitting objects, and the rest are discarded. As can be seen from Fig. 2 A, this rule applies to sourness, bitterness and astringency, so the TIRT coefficient K and R² can be directly obtained (k < 0, R² greater than 0.800). However, the intensity of salivary secretion and aftertaste-sweetness increased first and then decreased. Therefore, they have two K values. K_AS1 and K_SS1 represent the rising stage of taste, and K_AS2 and K_SS2 correspond to the falling stage (Table. 3). Finally, Multiply the above-mentioned sensory TIRT coefficient K by the sum of various sensory intensities (SI) at each time point to obtain the comprehensive taste index T of each taste. The aftertaste-sweetness and saliva secretion are slightly complex. K_AS1 and K_SS1 is multiplied by the sum of taste rising stages, K_AS2 and K_SS2 is multiplied by the sum of taste falling stages. The two are added to get the T value. The calculation formula is as follows: $$\mathrm{B}\mathrm{i}\mathrm{t}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{n}\mathrm{e}\mathrm{s}\mathrm{s},\mathrm{a}\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{c}\mathrm{y},\mathrm{s}\mathrm{o}\mathrm{u}\mathrm{r}\mathrm{n}\mathrm{e}\mathrm{s}\mathrm{s}:T=\sum _1^{n}SI\times K_{A,B,S}$$ $$\mathrm{S}\mathrm{w}\mathrm{e}\mathrm{e}\mathrm{t}\mathrm{n}\mathrm{e}\mathrm{s}\mathrm{s}\mathrm{a}\mathrm{n}\mathrm{d}\mathrm{s}\mathrm{a}\mathrm{l}\mathrm{i}\mathrm{v}\mathrm{a}\mathrm{s}\mathrm{e}\mathrm{c}\mathrm{r}\mathrm{e}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}:T=\sum _1^{n1}SI1\times K_1+\sum _{n1}^{n2}SI2\times K_2$$

n is the number of time points in the evaluation process; SI₁, n₁ represents the rising stage of taste; SI₂, n₂ is the falling stage. After calculating the T values of 5 taste senses of 12 PE batches, cluster analysis and PCA analysis were performed, and the results are shown in Fig. 2 B. According to the PCA results (Fig. 2 A R² = 0.804), cultivated and wild PE can be clearly distinguished on the comprehensive taste index, indicating that the model has better discriminative ability. Due to the large difference in the wild environment, it can be clearly found from the PCA result that the dispersion degree of the comprehensive taste index of wild PE is significantly greater than that of the cultivated PE, which indicates that the internal flavor quality of the samples is quite different. In addition, according to the loading scatter plot (Fig. 2 C), cultivated PE is most affected by the aftertaste sweetness, while wild PE has strong bitterness, acidity and astringency, which shows that the aftertaste sweetness intensity is an important factor to distinguish their taste. As the unique flavor of PE, aftertaste sweetness is widely welcomed by consumers. Many related beverages use it as a means of sales promotion. From the results, the reason why cultivated PE can be widely popular also lies in its stronger effect of returning sweet. From the cluster heat map of the comprehensive taste index in Fig. 2 D, it can be found that wild and cultivated can be divided into two groups, further indicating that the model has good discrimination ability. The data will be further correlated with key compounds to screen potential compounds that affect the overall taste of PE.

3.2 HS-SPME/GC-QQQ-MS/MS result

The unique aroma of PE is one of its sensory characteristics. Experienced suppliers will judge its quality according to the strength of its aroma. At present, there is no research on the chemical nature of PE aroma. Based on HS-SPME/GC-QQQ-MS/MS odor analysis technology, combined with retention time, parent ion and MRM monitoring ion pair and built in calibration curve, it can accurately and rapidly identify 150 odorants and analyze their concentration. This paper established a rapid method to identify the chemical components in the special odor of PE, and explored the key differences in the odor components of different types of PE.

Odor threshold refers to the minimum concentration of substances causing human olfaction (pg/mg). The performance of the odor components in PE is not entirely determined by the content, but also the threshold of the components. The threshold indicates the lowest concentration at which the human body can smell the substance. The ratio of the concentration to the threshold is called the odor activity values (OAV): $$\mathrm{O}\mathrm{V}\mathrm{A}=\frac{F}{M\ast T}$$

In the formula: OAV is the odor component intensity; F represents the content of the component (pg); M represents sample mass(mg); T represents the odor threshold of the component (pg/mg). Generally, the components with OAV value greater than 0.1 should be regarded as the components with obvious contribution to their odor, which can affect the flavor of samples in varying degrees. According to the above formula, all batches of PE lyophilized-dried powder were analyzed, and 18 components with OAV greater than 0.1 were found. Their content and OAV are listed in Table 4 and Table 5 respectively.

PCA analysis showed that the odor components of wild and cultivated PE could be clearly distinguished (Fig. 2 E). The OAV values of 18 kinds of odor components were compared in pairs (Fig. 2 F). When OAV ≥ 1, it indicates that it is a key odor substance, while 0.1 ≤ OAV < 1 indicates that it plays an important role in changing the overall odor of the sample. The difference components were screened by the following principles: 1. The average OAV value of odor was greater than 0.1; 2. The OAV of odor component was significantly higher than that of other groups; 3. The average value of group F was significantly different from that of group M. Based on the above principles, it was found that No.4–9 and No.13–15 compositions are the substances with the highest odor contribution and the maximum difference in two groups. According to the OVA, it can be divided into the Main and second difference odor components (Fig. 2 F).

Although hundreds of compounds have been identified in PE, only a few compounds actually contribute to sensory of PE flavor. From the results of Fig. 2 F, most of the aroma components are pleasant in cultivated PE, and the OAV is higher than wild PE. For example, trans-2-heptenal has a greasy, fresh, and fruity fragrance, and decanal has an orange peel fragrance. But cultivated PE also mixed with some bad smell, such as dimethyl trisulfide, which described as smelling like cabbage. It should be noted that the OAV of compound 8 in wild PE is much higher than that in cultivated PE. Compound 8, 2-Isobutyl-3-methoxypyrazine (IBMP) has a strong aroma of soil, spices and green pepper, which has a great impact on the overall flavor of PE. IBMP is very common in the food industry, especially in wine and beverage, which will have an adverse effect on the overall flavor of the product (Ling, Zhou, & Lan, 2021). IBMP accumulates during the early growth of fruits, and the content will be affected by photodegradation after the maturity period and the content will decrease rapidly (Sidhu, Lund, Kotseridis, & Saucier, 2015). In the process of fruit ripening, external factors such as climate, environmental conditions and canopy dressing will affect the light and temperature conditions, thus affecting the content of IBMP in the fruit (Gregan et al., 2012); Pickering, Karthik, Inglis, Sears, & Ker, 2008). For example, high temperature may cause the accumulation of IBMP in PE (Lei, Xie, Guan, Song, Zhang, & Meng, 2018). Artificial cultivation and climate change may be an important reason for the great difference of IBMP content between wild and cultivated PE. Therefore, IBMP may be a potential quality marker to distinguish wild PE from cultivated PE. Dimethyl trisulfide, another odor substance with high content in cultivated PE, has a certain meat flavor, which is not conducive to the flavor of PE. According to previous reports, dimethyl trisulfide may come from Strecker degradation of cysteine and methionine (Cho, Roman, Yeboah, & Konishi, 2007). In addition, No. 13, No. 14 and No. 15 compounds have a low contribution rate, but they can change the overall smell of PE and give it a unique aroma. The combination of these odor components makes the overall smell of PE very special, and some people describe cultivated PE as having an apple-like aroma. In short, the smell of cultivated PE is more pleasant. In brief, the smell of cultivated PE is more likely to be pleasant.

3.3 Mass imaging result

MS imaging technology provides a visual method for the characterization of metabolites in PE tissue, which can deeply reveal the component distribution and metabolic differences between wild and cultivated PE from the level of spatial distribution, and provide valuable information for the inner quality and the study of metabolic differences. In the experiment, the compositions were identified based on the literature and HMDB database (Yang, Kortesniemi, Liu, Karonen, & Salminen, 2012). Table.6 shows the main compounds identified in the MS imaging results (Cultivated PE on the left).

3.3.1

3.3.1 Distribution characteristics of organic acids

Organic acids are another kind of important secondary metabolites in plants. They play an important role in plant energy metabolism and plant response to external stresses. For example, malic acid is widely involved in photosynthesis and respiration of plants; citric acid, a common plant organic acid, has biological activities such as oxidation resistance and antibacterial activity (Chang, Foo, Loh, Lim, & Abdul Mutalib, 2020). In the mass imaging experiment, 9 organic acids and their distribution characteristics were identified. Among them, aconitic acid, citric acid, malic acid and fumaric acid are the typical components involved in the tricarboxylic acid cycle. As shown in Fig. 3, the contents of all 6 organic acids content are lower in the core and fruit compartment diaphragm (the obvious black lines in the fruit in Fig. 3), but increased significantly near the epidermis. This suggests that the metabolism of tricarboxylic acid in PE may be more vigorous in the tissue structure close to the epidermis. In particular, citric acid and malic acid are the most significant, their content in the epidermis is extremely high, which makes the sour substances preferentially released during the chewing process, causing their sour taste to be prominent. In addition, it is not difficult to find that the content of citric acid and malic acid in this two PEs is significantly different. The content of cultivated PE is lower and even distribution. This is one of the main reasons why the wild tastes extremely sour and difficult to eat.

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Fig. 3

Distribution characteristics of main organic acids in PE tissue.

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3.3.2

3.3.2 Distribution characteristics of Tannin and Phenolic acid

Tannin is the second major component in PE fresh fruits, and its highest content can reach 30%-40% of its dry weight (Huang, Ran, et al., 2021), which has strong antioxidant, anti-tumor, antibacterial and anti-hyperglycemic activities (Variya, Bakrania, & Patel, 2016). Like organic acids, they are mainly distributed near the epidermis. From Fig. 4, the distribution of tannins in the two PEs are obviously different, such as ellagic acid, malic acid gallate, mucic acid lactone gallate and galloylglucose.

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Fig. 4

Distribution characteristics of tannin and phenolic acid.

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Tannins are important secondary metabolites produced by plants in response to the growth environment. Wild PE grows in a harsh environment of high altitude, drought, heat, and less rain; while-cultivated PE generally grows in a warm and humid low-altitude environment. In the hot and dry environment, plants produce a lot of tannins as defense substances to resist external invasion and prevent the loss of water and other nutrients. Under dry heat stress, plants will promote phenylpropane biosynthesis and flavonoid biosynthesis, and make polyphenols and flavonoids accumulate. They can effectively resist the damage caused by dry heat and ultraviolet radiation, which is the performance of plants against external stress (Kumar et al., 2016). The wild PE epidermis is directly affected by solar radiation and drought, leading to more active metabolism and more accumulation of metabolites. This may explain why it is highly distributed in epidermis (Chen, Liu, Cui, Lu, Wang, Wu, et al., 2018; Morris, Carter, Hauck, Lanot, Theodorou, & Allison, 2021).

3.3.3

3.3.3 Distribution characteristics of nutrients

The nutrition of PE is one of the important properties of its edible value. It not only has high health benefits, but also affects its taste. In the experiment, 8 kinds of nutrients were identified (Fig. 5), including saccharides, vitamins, amino acids and fatty acids. It can be clearly seen from Fig. 5 that the nutrient content of cultivated PE is generally higher, indicating that it is rich in nutrients and more suitable for consumption. For example, the content of vitamins and sugars is higher, which can make the sweetness and aftertaste-sweetness of cultivated PE better. Surprisingly, the distribution of suspected fatty acids was found in the epidermis, which was usually found in the seed nucleus. Based on the above results, most of the components of PE are higher in the part near the epidermis, and from the perspective of the spatial distribution and content of the components, it explains the generation of the taste level of PE and the reason why cultivated PE tastes better than wild PE.

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Fig. 5

Distribution characteristics of nutrients.

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3.4 Metabolomics and taste correlation analysis

In order to further reveal the chemical differences and biological background between wild and cultivated PE, metabonomics method based on UPLC-QTOF-MS was used to study the main differences of their metabolic pathways. The quality control samples (QCS) were prepared by mixing the reference materials. QC samples gathered together showed that the repeatability of the system was good, and the data can be further studied. Through OPLS-DA analysis, it was found that there were significant differences in chemical composition and metabolites between group F and group M in negative ion mode ((Fig. 6 A R²Y = 0.99, Q² = 0.73). To explore the potential variables, S-plot was carried out. In S-plot (Fig. 6 B), the farther the variable deviates from the origin, the higher the value of the variable importance plot obtains. According to the accurate m/z and fragment characteristics (Ion fragment prediction matching) of the ion characteristic metabolites, and compared with the reference substance, a total of 33 characteristic metabolites were identified in the sample (Fig. 6 G). The results showed that the contents of tannins and flavonoids in wild PE were significantly higher than those in cultivated PE, which may be an important reason for the taste difference. Furthermore, the differential marker data were imported into metaboanalyst 5.0 for pathway enrichment (Fig. 6 C).

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Fig. 6

Results of OPLS-DA(A), S-plot(B), pathway enrichment analysis bubble plot (C), antioxidant activities (D), anti-hyperglycemic activities (E), antibacterial activity (F), heat maps of differential markers (G).

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The difference markers were marked as HMDB-ID, and imported into MetaboAnalyst5.0 for pathway analysis. The result (Fig. 6 C) shows that the selected metabolites mainly involve flavone and flavonol biosynthesis, flavonoid biosynthesis, histidine metabolism, arginine biosynthesis, pentose phosphate pathway, carbon fixation in photosynthetic organisms, pyrimidine metabolism, cysteine and methionine metabolism. Among them, the enrichment pathway with p < 0.05 was the flavone and flavonol biosynthesis, and flavonoid biosynthesis. Obviously, the generation of this metabolic difference is closely related to its different planting geographical environment. The results showed that the accumulation of phenolic metabolites of wild PE was significantly higher than that of cultivated PE, and the former grew in drought. Phenolic accumulation is very crucial to counteract the negative impacts of drought stress in plants(Ali, Ganai, Kamili, Bhat, Mir, Bhat, et al., 2018). Transcriptomic and metabolomic studies carried out on Arabidopsis plants confirmed that increased flavonoid accumulation under drought stress is very helpful to provide resistance. Accumulation and Biosynthesis of flavonols were also stimulated in plants under water deficit conditions accompanied by enhanced resistance against drought stress (Nakabayashi, Yonekura-Sakakibara, Urano, Suzuki, Yamada, Nishizawa, et al., 2014). For example, contents of flavonoids like kaempferol and quercetin were enhanced in tomato plants accompanied by enhanced drought tolerance (M. Li, Li, Zhang, Li, Gao, Ai, et al., 2018; Sánchez-Rodríguez, Moreno, Ferreres, Rubio-Wilhelmi Mdel, & Ruiz, 2011).

It is well known that plants growing in high altitude areas accumulate more flavonoid phenolics than plants in temperate regions. Under high light/UV radiation, the enhancement of flavonoid accumulation is due to the stimulated flavonoid biosynthesis pathway and its corresponding gene transcription level (Sharma, Shahzad, & Rehman, 2019). Flavonoids also act as light screens due to their capability of absorbing both visible (anthocyanins) and UV radiations (anthocyanins and colorless flavonoids), hence protecting plants from these harmful radiations (Agati, Brunetti, Di Ferdinando, Ferrini, Pollastri, & Tattini, 2013). This also explains why mass spectrometry imaging shows higher levels of secondary metabolites near the epidermis of wild PE. However, the enhanced synthesis of flavonoids and flavanols will increase their bitterness and worsen the taste, which is a phenomenon that should be avoided in beverages.

In order to further clarify the relationship between different compounds and taste, reveal the chemical properties of their taste, and provide a reference for quality control, this paper explored the correlation between differential markers and comprehensive taste index, and initially screens some quality markers that affect taste. Pearson correlation analysis was used in the data processing. It can be found that bitterness substances are closely related to tannins and flavonoids, such as quercetin (0.62), amlaic acid (0.76) and kaempferol (0.62). This further illustrates that the difference in biosynthesis of flavonoids and flavanols is the cause of the difference in flavor. The sourness and astringency are related to tannins such as sanguiin H2 (0.67) and amlaic acid (0.64). The formation of aftertaste sweetness is more complicated, and it has been reported that it is related to the catechins EC and EGC (Zhang, Yin, Chen, Wang, Du, Jiang, et al., 2016). But at present, the findings in this paper are related to some amino acid derivatives and glycosides, such as hesperetin 5,7-O-diglucuronide (0.64) and N-Acetyl-L -glutamate 5-semialdehyde (0.84). It is worth noting that amlaic acid is relevant to all bad tastes, which may be an important potential flavor quality marker. In addition, quercetin and related derivatives can also be used as quality markers that affect flavor.

3.5 Activity evaluation results

As a dietary supplement and functional beverage, PE has higher health benefits, which is due to its strong antioxidant activity and hypoglycemic effect. The anti-hyperglycemic activity of PE is mainly manifested in its anti-hyperglycemic activity α Inhibition of glucosidase (Majeed, Majeed, & Mundkur, 2020). Therefore, the antioxidant and anti-hyperglycemic activities of two PEs were determined respectively to clarify their difference in activity. As shown in Fig. 6 D, due to the different content of polyphenols, flavonoids and organic acids, the antioxidant activity of wild PE was significantly stronger than cultivated PE (p < 0.05). And surprisingly, both wild and cultivated PE in Fig. 6.E showed strong anti-hyperglycemia ability compared with the positive group. Although there are certain differences in activity within the cultivated group, there is no significant difference between the wild and cultivated groups. The antibacterial experiment uses the ultrasonic extract of freeze-dried powder (12 times water, commonly used amount) as the sample solution, which is the commonly used extraction dose of PE in the beverage industry. The results (Fig. 6 F) showed that the antibacterial activity of wild PE was significantly stronger than cultivated PE, and the latter had almost no other antibacterial effect except Aspergillus flavus. This may be closely related to the accumulation of secondary metabolites such as flavonoids and tannins. In addition, because the experiment used a water extract, which made many lipophilic tannins and flavonoids with strong antibacterial activity difficult to dissolve in it, limiting their biological activity (Liu, Zhao, Luo, Yang, & Jiang, 2009). Based on the above results, wild PE has better biological activity.