Phytochemical Analysis, Antioxidant and Enzyme-Inhibitory Activities, and Multivariate Analysis of Insect Gall Extracts of Picea koraiensis Nakai

Picea koraiensis Nakai (PK) is an evergreen tree. It plays an important role in landscaping and road greening. Insect galls of PK are formed by parasitism of the adelgid Adelges laricis. Except for phenolics, other chemical constituents and biological activity of insect gall from PK are still unknown. Thus, here, we performed phytochemical and biological activity analyses of PK insect gall extracts, aiming to turn waste into treasure and serve human health. PK insect gall extracts were prepared using seven solvents. Antioxidant activities of the extracts were examined via antioxidant assays (radical and oxidizing substance quenching, metal chelating, and reducing power). The inhibitory activities of the extracts were determined toward the key human-disease-related enzymes α-glucosidase, α-amylase, cholinesterase, tyrosinase, urease, and xanthine oxidase. The content of numerous active constituents was high in the methanol and ethanol extracts of PK insect gall, and these extracts had the highest antioxidant and enzyme-inhibitory activities. They also showed excellent stability and low toxicity. These extracts have potential for use as stabilizers of olive and sunflower seed oils.


Introduction
Bulged parts or fruit-like structures are common on the leaves and branches of some plants. People often mistakenly think these are the fruit or seed of the plant, but they are not-these structures are called "galls". Galls are nodules or protuberances formed by accelerating cell division and abnormal differentiation after plant tissue is stimulated by another organism. There are >2000 gall-forming organisms; gall-forming animals include insects, mites, and nematodes, while gall-forming microorganisms include bacteria, fungi, and viruses. Galls can be produced in almost all kinds of higher plants. The phenomenon of insect galls-which are galls formed after plant tissue is stimulated by feeding or oviposition by insects [1]-is very common.
Insect gall is a product of parasitic interaction between insects and plants. Insects are the beneficiary: they obtain food and a habitat from the host plant, and they can avoid predation and adverse climates. Plants are the victims of this interaction: plant growth is slowed, and plant height and leaf area are decreased. Furthermore, serious cases can cause death [2]. However, in most cases, the damage of galls to plants is limited, and the formation of plant galls is an act of self-protection to some extent. As an abnormal proliferative tissue, the content of chemical components, such as fat, protein, starch, and tannic acid, in galls is much higher than in normal plant tissues. death [2]. However, in most cases, the damage of galls to plants is limited, and the formation of plant galls is an act of self-protection to some extent. As an abnormal proliferative tissue, the content of chemical components, such as fat, protein, starch, and tannic acid, in galls is much higher than in normal plant tissues.
Galla chinensis are insect galls formed on the Chinese sumac tree Rhus chinensis; these galls are used extensively in Chinese traditional medicine because their tannin content is as high as 70-80%. Tannic acid is extracted from these galls, and its processed products are gallic acid and pyrogallic acid, which have antidiarrheal, antiperspirant, hemostatic, and wound healing effects. A decoction of Galla chinensis can be used to treat peptic ulcers and inflammation [3], and it is also used in the adjuvant treatment of diabetes mellitus and cancer [4].
Picea koraiensis Nakai (PK) is an evergreen tree in the family Pinaceae. It is distributed in the northeast Xiao Hinggan Mountains and the Jilin Mountains of China, the Russian Far East, and North Korea. It is a precious tree species that plays an important role in landscaping and road greening. Insect galls of PK endanger the shoots and buds of the tree and affect its normal growth and development, and the damaged branches dry up and die. Insect galls of PK are formed by the parasitism of Adelges laricis. Larix kaempferi is a secondary host in the same area [5]. The shoots and buds stimulated by the adelgid gradually grow and form spherical insect galls. The interior of the gall is composed of multiple cavities, in which there are several to dozens of Adelges laricis. There are "fish scale" patterns on the surface of the galls (Figure 1). The pattern is light pink at first, turns purple-red with the growth of the gall, and finally turns black. At the end of autumn, the pattern cracks, and the Adelges laricis drill out. On Larix kaempferi, Adelges laricis suck the sap from needles and shoots and produce large amounts of moldy branches and waxy secretions that dry; the growth of the tree is severely affected [5]. The control method is to avoid mixed or close planting of PK and to manually remove gall from PK. Removed galls are usually thrown away as waste. In recent years, some researchers have found that phenolics are usually produced in cones as well as in galls from PK in response to insect-induced tissue damage [6,7]. Except for phenolics, other chemical constituents and biological activity of insect gall from PK are, however, unknown. Thus, here, we performed a phytochemical analysis and assessed the biological activities of insect gall extracts from PK, aiming to turn waste into treasure and serve human health. In recent years, some researchers have found that phenolics are usually produced in cones as well as in galls from PK in response to insect-induced tissue damage [6,7]. Except for phenolics, other chemical constituents and biological activity of insect gall from PK are, however, unknown. Thus, here, we performed a phytochemical analysis and assessed the biological activities of insect gall extracts from PK, aiming to turn waste into treasure and serve human health.

Qualitative Phytochemical Analysis
Eleven classes of phytochemicals were identified in PK insect gall (Table 1). Saponins, anthraquinones, cardiac glycosides, and cyanogenic glycosides were not detected.

Yields
Seven solvents with distinctive polarities were selected to extract PK insect gall powder. The extraction yields ranged from 5.28 ± 0.21% to 34.32 ± 0.41% (w/w) ( Table 2). The aqueous extract had the highest yield because of the abundance of water-soluble constituents (such as polyphenols, phenolics, polysaccharides, and others), followed by the methanol and ethanol extracts. Hexane extract had the lowest yield. Carbohydrates are important constituents of animal and plant cells. They have physiological functions, such as storing and supplying energy, saving protein, and resisting the production of ketone bodies [8]. Glycosides also have various pharmacological effects, which could be used to treat neuronal diseases [9].
The total carbohydrate content (TCC) was detected in various solvent extracts of PK insect gall ( Table 3). The TCC of the solvent extracts was 5.42 ± 0.31 to 325.83 ± 0.47 mg glucose equivalents (GE)/g extract. Significant differences were found among the groups: the methanol extract had the highest TCC, followed by the aqueous extract. The hexane extract had the lowest TCC. This may be because many components in the methanol extract are glycosides.

Total Protein Content (TP ro C)
Plant protein has a wide range of properties and nutritional value, and it is easily digested and absorbed by the human body. It has various functions, such as immune regulation, and antioxidation and antifatigue activities [10]. Only the aqueous extract of PK insect gall contained protein, which was 313.32 ± 11.61 mg bovine serum albumin equivalents (BSAE)/g extract (Table 3).

Total Triterpenoid Content
Triterpenoids have a variety of biological activities, such as anticancer, antiallergy, antiatherosclerosis, and antiulcer properties [11]. The total triterpenoid content (TT ri C) of the seven different solvent extracts of PK insect gall was determined (Table 3). Triterpenoid was not detected in the aqueous extract. The TT ri C of the other solvent extracts was 0.29 ± 0.03 to 1.89 ± 0.10 mg ginsenoside Re equivalents (GRE)/g extract. The highest content was in the hexane extract, followed by the dichloromethane extract. The lowest content was in the ethanol extract.

Total Phenolic Content
Plant polyphenols are a class of active substances with good antioxidant capacity. They have antiviral and antitumor functions, and they are active in the prevention of cardiovascular disease and dementia [12]. They are widely used in the fields of cosmetics, food, and medicine [13]. The total phenolic content (TP he C) was determined in the seven solvent extracts of PK insect gall ( Table 3). The TP he C ranged from 0.92 ± 0.03 to 77.11 ± 0.52 mg GAE/g extract. TP he C was strongly correlated with solvent polarity. Table 3. Total carbohydrate content (TCC), total protein content (TP ro C), total triterpenoid content (TT ri C), total phenolic content (TP he C), total flavonoid content (TFC), total phenolic acid content (TPAC), gallotannin content (GC), condensed tannin content (CTC), and total tannin content (TT an C) of insect gall of Picea koraiensis extracted with different solvents.

Total Flavonoid Content
Flavonoids can prevent cell degeneration and aging, inhibit the growth of cancer cells, and control blood pressure and cholesterol; they also have good preventive effects against cardiovascular and cerebrovascular diseases [14]. The total flavonoid content (TFC) in different solvent extracts of PK insect gall was low (Table 3), ranging from 2.42 ± 0.22 to 7.52 ± 0.61 mg quercetin equivalents (QE)/g extract. The highest content was in the acetone extract, followed by the ethanol extract (there was no significant difference between the two).

Total Phenolic Acid Content
The total phenolic acid content (TPAC) was determined in the seven solvent extracts ( Table 3). The content range was 1.38 ± 0.21 to 39.92 ± 2.54 mg caffeic acid equivalents (CAE)/g extract. The content was highest in the ethanol extract, followed by the methanol extract; it was lowest in the dichloromethane extract. Similarly to TP he C, TPAC was strongly correlated with solvent polarity.

Total Tannin Content, Gallotannin Content, and Condensed Tannin Content
Tannin has antibacterial and antiviral effects, and it can also be used as an antidote for alkaloid and heavy metal poisoning. It has strong reducing properties, and it can remove superoxide free radicals in the body and delay aging [15].
The gallotannin content (GC), condensed tannin content (CTC), and total tannin content (TT an C) of the solvent extracts of PK insect gall were determined (Table 4); these ranged from 0.36 ± 0.01 to 1.81 ± 0.22, 2.22 ± 0.23 to 38.54 ± 0.23, and from 4.32 ± 0.01 to 53.64 ± 0.22 mg CAE/g extract, respectively. The methanol extract had the highest GC and CTC, followed by the ethanol extract. The ethanol extract had the highest TT an C, followed by the methanol extract. DPPH and ABTS scavenging assays are the most common methods for detecting free radical scavenging ability in vitro [16]. DPPH is a fat-soluble free radical and ABTS is a water-soluble free radical. The type of antioxidant can be judged by the difference in the scavenging activity of the antioxidant toward these two test compounds.
In experiments with DPPH, the methanol and ethanol extracts of PK insect gall showed strong antioxidant capacity (Table 4), and both were higher than butylated hydroxytoluene (BHT). In experiments with ABTS, the ethanol and methanol extracts again had high free radical scavenging ability.

Hydroxyl Radicals, Superoxide Radicals, and Singlet Oxygen
Hydroxyl radicals, superoxide anions, and singlet oxygen are all produced in the body and result in oxidative toxicity to cells. Scavenging of these radicals reflects the capacity of antioxidants [17].
The hexane extract of PK insect gall had the highest hydroxyl radical scavenging activity, followed by the ethyl acetate and dichloromethane extracts. In superoxide anion scavenging experiments, the ethyl acetate extract had the strongest scavenging activity, while the methanol and ethanol extracts had no scavenging activity. Three extracts (water, methanol, and ethanol) had weak singlet oxygen scavenging activity (Table 4).

FRAP and CUPRAC
The antioxidant capacity of samples can also be determined by measuring their ability to reduce iron and copper ions [18]. FRAP experiments were performed in acidic conditions, and CUPRAC experiments were performed in neutral conditions; the latter method is closer to the physiological environment, and, thus, the results can be considered more accurate.
In FRAP experiments, the methanol extract of PK insect gall had the highest antioxidant capacity, followed by the ethanol extract. They were indistinguishable from BHT in reducing ability. The results of CUPRAC experiments were consistent with those of FRAP experiments (Table 5).

Metal Chelating
The presence of iron and copper ions accelerates the Fenton reaction and aggravates oxidative stress in vivo [19]. Therefore, the discovery of effective metal ion chelators is of great significance.
The ferrous-ion-chelating activity of the hexane extract of PK insect gall was highest, followed by the acetone and dichloromethane extracts. The copper-ion-chelating activity of the aqueous extract was the highest, followed by the ethanol and methanol extracts ( the PK insect gall extracts, the ethanol extract had the greatest H 2 O 2 scavenging activity, followed by the aqueous and acetone extracts ( Table 6).

β-Carotene Bleaching
β-Carotene is a polyene colorant that is easily oxidized, causing it to lose its yellow color [21]. In the presence of antioxidants in solution, the bleaching rate of β-carotene is slowed; as the capacity of an antioxidant becomes stronger, the decrease in β-carotene absorbance becomes slower. In experiments using the solvent extracts of PK insect gall with β-carotene, the acetone extract had the highest antioxidant capacity, followed by the dichloromethane and ethyl acetate extracts. There was no statistical difference among the three, and their antioxidant capacity was close to those of BHT and butyl hydroxyanisole (BHA). The aqueous extract had the weakest preventive effect on β-carotene bleaching ( Figure 2, Table 6).

Hypochlorous Acid
Hypochlorous acid (HClO) is an endogenous strong oxidant. It helps defend against pathogen invasion. However, excess HClO disrupts the oxidative balance of the organism, leading to the occurrence of disease [22]. Among the extracts of PK insect gall, only the

Hypochlorous Acid
Hypochlorous acid (HClO) is an endogenous strong oxidant. It helps defend against pathogen invasion. However, excess HClO disrupts the oxidative balance of the organism, leading to the occurrence of disease [22]. Among the extracts of PK insect gall, only the methanol, ethanol, and aqueous extracts had the ability to scavenge HClO (Table 6).

Nitric Oxide
Nitric oxide (NO) is a colorless, slightly water-soluble, fat-soluble gas. It can pass through biofilms freely in the living body. Therefore, it can participate in many biological activities and pathological and physiological processes. By acting on target molecules, NO can modify protein function and cause cellular damage. The absorbance of NO increased with time ( Figure 3). The methanol extract of PK insect gall showed the best NO scavenging ability.

Hypochlorous Acid
Hypochlorous acid (HClO) is an endogenous strong oxidant. It helps defend aga pathogen invasion. However, excess HClO disrupts the oxidative balance of the organ leading to the occurrence of disease [22]. Among the extracts of PK insect gall, only methanol, ethanol, and aqueous extracts had the ability to scavenge HClO (Table 6).

Nitric Oxide
Nitric oxide (NO) is a colorless, slightly water-soluble, fat-soluble gas. It can p through biofilms freely in the living body. Therefore, it can participate in many biolog activities and pathological and physiological processes. By acting on target molecules, can modify protein function and cause cellular damage. The absorbance of NO increa with time ( Figure 3). The methanol extract of PK insect gall showed the best NO scave ing ability.

α-Amylase and α-Glucosidase
Diabetes has become one of the major diseases that threaten human health. α-glucosidase and α-amylase promote the breakdown of food in the oral cavity and gastrointestinal tract, releasing glucose into the blood and therefore increasing blood sugar [23]. Inhibitors of these enzymes are the first choice for treatment of diabetes, but they have significant side effects. Therefore, it would be preferable to find safe inhibitors of these enzymes from natural products.
The α-glucosidase inhibitory activity of the methanol extract of PK insect gall was high and better than that of acarbose, which deserves further study. The α-amylase inhibitory activity of the acetone extract was excellent, and there was no significant difference compared with acarbose (Table 7).

AChE and BChE
Alzheimer's disease has a complex pathology [24]. Functional deficit of the cholinesterergic system is closely related to Alzheimer's disease; cholinesterase inhibitors are the main direction of anti-Alzheimer's disease drug research. The aqueous and methanol extracts of PK insect gall had AChE inhibitory activity, and the methanol extract also had BChE inhibitory activity (Table 7).

Tyrosinase
Tyrosinase has important physiological functions. Its abnormal expression can cause melanoma and early-onset AD. Tyrosinase inhibitors have whitening functions and can be used as cosmetic additives [25]. The dichloromethane, methanol, and ethanol extracts of PK insect gall showed notable inhibitory activity toward tyrosinase (Table 7).

Xanthine Oxidase
The main function of xanthine oxidase is to catalyze the oxidation of hypoxanthine to xanthine, which is further oxidized to uric acid. Too much uric acid in the body can lead to hyperuricemia and gout [26]. Inhibition of xanthine oxidase activity is thus a good target for the clinical treatment of hyperuricemia and gout. The acetone extract of PK insect gall had the highest xanthine oxidase inhibitory activity, followed by the methanol extract (Table 7).

Urease
Urease, produced by Helicobacter pylori, catalyzes the hydrolysis of urea and increases gastric pH. This leads to the occurrence of diseases, such as gastric ulcers and atrophic gastritis. Urease inhibitors interfere with the binding of urea to the catalytic site of the enzyme, thereby decreasing its catalytic activity and reducing the harm caused by Helicobacter pylori infection [27]. Ethyl acetate, acetone, and hexane extracts of PK insect gall showed greater urease inhibitory activity than thiourea (Table 7), which deserves further in-depth study.

UHPLC-MS Analysis
The content of active ingredients in the methanol extract of PK insect gall was high, and the biological activity was good. Therefore, it was selected for further stability studies. We investigated the chemical composition of the methanol extract of PK insect gall using UHPLC-ESI-Q-TOF-MS. Twenty-five bioactive substances were identified by matching molecular ions and fragment ions with reference data ( Table 8). The structures of these compounds are shown in  molecular ions and fragment ions with reference data ( Table 8). The structures of these compounds are shown in   In the UHPLC-MS results, peak 1 had a [M + H] + ion at m/z 337.1723, and its main fragment ion was at m/z 251.0314 ([M − C3H8N3] + ) and was identified as N2-fructopyranosylarginine [28]. Peak 2 at m/z 104.1069 was tentatively assigned as choline [29].  In the UHPLC-MS results, peak 1 had a [M + H] + ion at m/z 337.1723, and its main fragment ion was at m/z 251.0314 ([M − C 3 H 8 N 3 ] + ) and was identified as N2fructopyranosylarginine [28]. Peak 2 at m/z 104.1069 was tentatively assigned as choline [29]. Peak 3 had a [M + H] + ion at m/z 158.1538, and its main fragment ion was at m/z 140.1438 [M − NH 3 ] + . These results were characteristic of 4-(aminomethyl)-1-(diaminomethylene) piperidinium. Peak 4 had a [M + H] + peak at m/z 136.0621 that generated a main fragment ion at m/z 118.0859 [M − NH 3 ] + , which was characteristic of adenine [30]. The mass spectrum of peak 5 revealed an ion at m/z 140.1435. The MS 2 spectrum of this ion showed a fragment at m/z 98.0964. The main fragment ions of peak 6 (m/z 349.1260) appeared at m/z 344.1706 [M + NH 4 ] + , and this peak was assigned as citrusin C [31]. Peak 7 at m/z 545.1999 had MS 2 ions at m/z 540.2458 ([M + NH 4 ] + ) and 285.1132 ([C 13 H 17 O 7 ] + ), and was identified as isolariciresinol 4 -O-beta-D-glucoside [32]. Peak 8 revealed an ion at m/z 465.1027). A fragment ion at m/z 303.0507 originated from the ion at m/z 465.1027 and was related to hexose (162 Da) loss. These results indicated that peak 8 was gossypitrin [33]. The mass spectrum of peak 9 revealed an ion at m/z 369.1537. The MS 2 spectrum of this ion showed a fragment at m/z 167.1074. Peak 10 (m/z 449.1072) generated fragment ions at m/z 287.0551, which was related to loss of hexose. Thus, peak 10 was assigned to isoorientin [34].

Stability Studies of Methanol and Ethanol Extracts
The outcomes of stability studies of the methanol and ethanol extracts of PK insect gall are shown in Figures 6-8. The TP he C value and ABTS scavenging activity of the extracts were broadly stable with respect to adjustments in the pH. The TP he C of the extracts was highest at a pH of 1 and decreased slightly with increasing pH. This is because phenolic substances can dissolve better in acidic conditions and, in strongly alkaline conditions, some phenolic components are degraded [50]. The TP he C of the methanol and ethanol extracts decreased with heating time; indeed, the TP he C of the methanol extract decreased greatly (p < 0.05). The ABTS scavenging activity of both extracts decreased slightly with the prolongation of heating time. m/z 318.2156 ([M + H+Na] ), and was tentatively assigned as 18-hydroxy-9,11,13-octadecatrienoic acid [48]. Peak 30 (m/z 301.2160) generated fragment ions at m/z 183.1169 and 169.1012, which were related to loss of -C7H11, and -C8H13, respectively. Thus, peak 30 was assigned to α-linolenic acid [49].

Stability Studies of Methanol and Ethanol Extracts
The outcomes of stability studies of the methanol and ethanol extracts of PK insect gall are shown in Figures 6-8. The TPheC value and ABTS scavenging activity of the extracts were broadly stable with respect to adjustments in the pH. The TPheC of the extracts was highest at a pH of 1 and decreased slightly with increasing pH. This is because phenolic substances can dissolve better in acidic conditions and, in strongly alkaline conditions, some phenolic components are degraded [50]. The TPheC of the methanol and ethanol extracts decreased with heating time; indeed, the TPheC of the methanol extract decreased greatly (p < 0.05). The ABTS scavenging activity of both extracts decreased slightly with the prolongation of heating time.
In stability experiments using an in vitro simulation of the human digestive system, the TPheC value of the methanol and ethanol extracts of the PK insect gall decreased gradually with time. This may be related to the effects of gastric acid, pepsin, trypsin, pancreatin, and bile on the extracts. The ABTS scavenging ability of the extracts showed the same trend as the TPheC.

Oxidative Stability Studies of Oils
Adding antioxidants improves the stability of the oils, but there are risks and hazards with using synthetic antioxidants. Therefore, the use of safe antioxidants from natural sources is desirable. Figures 9 and 10 show that in K232 experiments, the addition of methanol extract of PK insect gall in large doses to extra virgin olive oil (EVOO) had good consequences superior to those of BHT and tertiary butylhydroquinone (TBHQ) compared with the methanol extract, with the ethanol extract having slightly lesser effects. The addition of the methanol or ethanol extracts to EVOO in K270 experiments showed the same trend, and their effects were similar to those of BHT. Figure 11 shows that the addition of methanol or ethanol extracts in large doses to cold-pressed sunflower oil (CPSO) was superior to the addition of BHT or TBHQ in peroxide value experiments. Figure 12 shows that in acidity values experiments, the addition of methanol extract in large doses to EVOO was effective, while the addition of methanol or ethanol extracts in small doses to CPSO was more effective. In stability experiments using an in vitro simulation of the human digestive system, the TP he C value of the methanol and ethanol extracts of the PK insect gall decreased gradually with time. This may be related to the effects of gastric acid, pepsin, trypsin, pancreatin, and bile on the extracts. The ABTS scavenging ability of the extracts showed the same trend as the TP he C.

Oxidative Stability Studies of Oils
Adding antioxidants improves the stability of the oils, but there are risks and hazards with using synthetic antioxidants. Therefore, the use of safe antioxidants from natural sources is desirable. Figures 9 and 10 show that in K 232 experiments, the addition of methanol extract of PK insect gall in large doses to extra virgin olive oil (EVOO) had good consequences superior to those of BHT and tertiary butylhydroquinone (TBHQ) compared with the methanol extract, with the ethanol extract having slightly lesser effects. The addition of the methanol or ethanol extracts to EVOO in K 270 experiments showed the same trend, and their effects were similar to those of BHT. Figure 11 shows that the addition of methanol or ethanol extracts in large doses to cold-pressed sunflower oil (CPSO) was superior to the addition of BHT or TBHQ in peroxide value experiments. Figure 12 shows that in acidity values experiments, the addition of methanol extract in large doses to EVOO was effective, while the addition of methanol or ethanol extracts in small doses to CPSO was more effective.

Cell Viability
The cellular morphology after TM 3 mouse Leydig cells were treated with methanol or ethanol extracts of PK insect gall for 24 and 48 h is shown in Figures S69 and S70. Overall, no cytotoxicity was observed for either the methanol or ethanol extracts after 24 h of incubation; however, the methanol extracts showed low-level toxicity after 48 h of incubation, while the ethanol extracts were comparably safe ( Table 9). The methanol and ethanol extracts of PK insect gall could perhaps be used as oil stabilizers.

Cell Viability
The cellular morphology after TM3 mouse Leydig cells were treated with methanol or ethanol extracts of PK insect gall for 24 and 48 h is shown in Figures S69 and S70. Overall, no cytotoxicity was observed for either the methanol or ethanol extracts after 24 h of incubation; however, the methanol extracts showed low-level toxicity after 48 h of incubation, while the ethanol extracts were comparably safe ( Table 9). The methanol and ethanol extracts of PK insect gall could perhaps be used as oil stabilizers.

Multivariate Analysis
Through analysis of the experimental outcomes, a good correlation was found between the TCC values and the antioxidant activity ( Figure 13). This implies that the carbohydrates in the methanol extract are flavonoid glycosides. Good correlations were observed between the TPheC, TTanC, TPAC, CTC, and GC values and the antioxidant activity and α-glucosidase and BChE inhibitory activities, as they were between the TTriC value and hydroxyl radical, iron chelating, β-carotene, and α-amylase inhibitory activities (Figure 13). Good correlations were observed between the TFC value and DPPH, ABTS, and

Multivariate Analysis
Through analysis of the experimental outcomes, a good correlation was found between the TCC values and the antioxidant activity ( Figure 13). This implies that the carbohydrates in the methanol extract are flavonoid glycosides. Good correlations were observed between the TP he C, TT an C, TPAC, CTC, and GC values and the antioxidant activity and α-glucosidase and BChE inhibitory activities, as they were between the TT ri C value and hydroxyl radical, iron chelating, β-carotene, and α-amylase inhibitory activities ( Figure 13). Good correlations were observed between the TFC value and DPPH, ABTS, and CUPRAC ( Figure 13). The α-glucosidase and BChE inhibitory activity was linked to antioxidant capacity ( Figure 14).
Univariate statistical methods were used, p-values < 0.05 were considered significant for all active constituents, and biological activities were assessed separately. A multiple comparative analysis applying least significant difference (LSD) post hoc analysis showed that some samples showed the same content and activity against some active constituents' content and biological activities. Therefore, the properties of the samples depended on their active constituents. The dataset was applied to the principal component analysis (PCA). The results are depicted in Figure 15.  (Figure 13). The α-glucosidase and BChE inhibitory activity was linked to antioxidant capacity (Figure 14).  Univariate statistical methods were used, p-values < 0.05 were considered significant for all active constituents, and biological activities were assessed separately. A multiple comparative analysis applying least significant difference (LSD) post hoc analysis showed  (Figure 13). The α-glucosidase and BChE inhibitory activity was linked to antioxidant capacity (Figure 14).  Univariate statistical methods were used, p-values < 0.05 were considered significant for all active constituents, and biological activities were assessed separately. A multiple comparative analysis applying least significant difference (LSD) post hoc analysis showed TTriC value and hydroxyl radical scavenging, iron-chelating, and tyrosinase-inhibitory activity. The second cluster, including the ethyl acetate and acetone extracts, had the highest TFC value, superoxide radical scavenging activity, and α-amylase, xanthine oxidase, and BChE inhibition, and it showed the greatest prevention of β-carotene bleaching. The third cluster, the aqueous extract, displayed the highest copper-chelating activity and AChE inhibition. The fourth cluster, composed of the methanol and ethanol extracts, exhibited high TCC, TPheC, TPAC, GC, CTC, and TTanC, high antioxidant activity, and high α-glucosidase and AChE inhibition.  Three principal components (PCs), explaining 85.4% of the data variance, were identified ( Figure 15D). The contributions of the active constituents and biological activities to the three PCs are expressed in Figure 15E. PC1 summarized 59.4% of the data variance and differentiated samples in accordance with active constituents' content and antioxidant capacities. The values for PC2 and PC3, representing other sources of variability (enzyme-inhibitory activity and TFC), were 15.4% and 10.6%, respectively. The analysis of factorial maps ( Figure 15A-C) implied that the methanol and ethanol extracts were distinct from the other extracts of PK insect gall. However, the resolution between the other samples was poor.
Accordingly, the PCA outcomes were applied for hierarchical clustering analysis. The analysis of the three PCs from PCA identified four clusters ( Figure 16). The first cluster, which comprised the dichloromethane and hexane extracts, displayed the highest TT ri C value and hydroxyl radical scavenging, iron-chelating, and tyrosinase-inhibitory activity. The second cluster, including the ethyl acetate and acetone extracts, had the highest TFC value, superoxide radical scavenging activity, and α-amylase, xanthine oxidase, and BChE inhibition, and it showed the greatest prevention of β-carotene bleaching. The third cluster, the aqueous extract, displayed the highest copper-chelating activity and AChE inhibition. The fourth cluster, composed of the methanol and ethanol extracts, exhibited high TCC, TP he C, TPAC, GC, CTC, and TT an C, high antioxidant activity, and high α-glucosidase and AChE inhibition.

Preparation of Different Extracts of PK Insect Gall for Quantitative Phytochemical Analysis and UHPLC-MS Analysis
The collected samples of PK insect gall were dried in a cool ventilated place and pulverized to powder. The powder (20 g) was added to a single-neck round-bottomed flask (glass, 500 mL), followed by the addition of 200 mL of various solvents (water, methanol, ethanol, acetone, ethyl acetate, dichloromethane, or hexane) and refluxing using a hotplate magnetic stirrer employing methyl silicone oil as the heating medium for 6 h at the respective boiling points of the solvents. The extracts were filtered through a Whatman No.1 filter paper and evaporated under reduced pressure at <50 • C until dry using a rotary evaporator. All dried extracts were weighed and stored at −20 • C until use. The yield was calculated as % yield = (weight of dry extract/initial weight of dry sample) × 100.

Qualitative Phytochemical Analysis
Qualitative phytochemical analysis of 15 types of chemical components was performed in accordance with our previous methods [51].

Quantitative Phytochemical Analysis
The TCC, TP ro C, TT ri C, TP he C, TFC, TPAC, TT an C, CTC, and GC were determined in accordance with our previous methods [51,52]; the specific experimental steps are as follows.

Determination of TCC
Briefly, 250 µL of extract of PK insect gall in distilled water, 125 µL of phenol solution (5%), and 625 µL of H 2 SO 4 were mixed in an Eppendorf tube and incubated for 30 min. Subsequently, 200 µL of the sample was pipetted from each Eppendorf tube onto a microplate. A calibration curve was produced based on glucose (0-200 mg/L) as a standard. The absorbance of the sample was recorded at 490 nm against a blank sample consisting of extract of PK insect gall with distilled water. The mean of three readings was used, and TCC was expressed in milligrams of GE/g of extract of PK insect gall.

Determination of TP ro C
Briefly, 200 µL of bicinchoninic acid working solution and 20 µL of extract of PK insect gall in distilled water were mixed in a microplate and incubated at 37 • C for 30 min. A calibration curve was produced based on bovine serum albumin (0-500 mg/L) as a standard. The absorbance of the sample was recorded at 562 nm against a blank sample consisting of extract of PK insect gall with distilled water. The mean of three readings was used, and TP ro C was expressed in milligrams of BSAE/g of extract of PK insect gall.

Determination of TT ri C
Briefly, 180 µL of extract of PK insect gall in acetic anhydride and 20 µL of H 2 SO 4 were mixed in a microplate and incubated at room temperature for 10 min. A calibration curve was produced based on ginsenoside Re (0-400 mg/L) as a standard. The absorbance of the sample was recorded at 350 nm against a blank sample consisting of extract of PK insect gall with acetic anhydride. The mean of three readings was used, and TT ri C was expressed in milligrams of GRE/g of extract of PK insect gall.

Determination of TP he C
Briefly, 100 µL of Folin and Ciocalteu's phenol reagent (FC reagent) (1 M) and 200 µL of extract of PK insect gall in distilled water were mixed in an Eppendorf tube and incubated for 5 min. Subsequently, 500 µL of Na 2 CO 3 solution (20%) was added and allowed to stand at room temperature for 40 min in the dark (with mixing every 10 min). Subsequently, 200 µL of the sample was pipetted from each Eppendorf tube onto a microplate. A calibration curve was produced based on gallic acid (0-100 mg/L) as a standard. The absorbance of the sample was recorded at 750 nm against a blank sample consisting of extract of PK insect gall with distilled water and Na 2 CO 3 . The mean of three readings was used, and TP he C was expressed in milligrams of GAE/g of extract of PK insect gall.

Determination of TFC
Briefly, 100 µL of AlCl 3 (2%) in methanol and 100 µL of extract of PK insect gall in methanol were mixed in a microplate and incubated at room temperature for 10 min. A calibration curve was produced based on quercetin (0-100 mg/L) as a standard. The absorbance of the sample was recorded at 415 nm against a blank sample consisting of extract of PK insect gall with methanol. The mean of three readings was used, and TFC was expressed in milligrams of QE/g of extract of PK insect gall.

Determination of TPAC
Briefly, 20 µL of extract of PK insect gall in distilled water, 20 µL of Arnow reagent, 20 µL of HCl solution (0.1 M), 120 µL of distilled water, and 20 µL of NaOH solution (1 M) were mixed in a microplate and recorded immediately at 490 nm against a blank sample (Arnow reagent was replaced with distilled water). A calibration curve was produced based on caffeic acid (0-100 mg/L) as a standard. The mean of three readings was used, and TPAC was expressed in milligrams of CAE/g of extract of PK insect gall.

Determination of TT an C
Briefly, 200 µL of FC reagent (1 M) and 200 µL of extract of PK insect gall in distilled water were mixed in an Eppendorf tube and incubated for 5 min. Subsequently, 100 µL of Na 2 CO 3 solution (20%) and 1500 µL of distilled water were added and allowed to stand at room temperature for 30 min in the dark (with mixing every 10 min). Subsequently, 200 µL of the sample was pipetted from each Eppendorf tube onto a microplate. A calibration curve was produced based on tannic acid (0-200 mg/L) as a standard. The absorbance of the sample was recorded at 725 nm against a blank sample consisting of extract of PK insect gall with distilled water and Na 2 CO 3 . The mean of three readings was used, and TT an C was expressed in milligrams of TAE/g of extract of PK insect gall.

Determination of GC
Briefly, 875 µL of extract of PK insect gall in methanol and 375 µL of saturated KIO 3 solution were mixed in an Eppendorf tube and incubated at 15 • C for 120 min. A calibration curve was produced based on gallic acid (0-400 mg/L) as a standard. The absorbance of the sample was recorded at 550 nm against a blank sample (KIO 3 was replaced with distilled water). The mean of three readings was used, and GC was expressed in milligrams of GAE/g of extract of PK insect gall.

Determination of CTC
Briefly, 4 mg of phloroglucinol was added to 2 mL of extract of PK insect gall in distilled water. Subsequently, 1 mL of HCl solution and 1 mL of formaldehyde solution were added and mixed in an Eppendorf tube and incubated at room temperature overnight. The precipitate was separated by filtration, and the unprecipitated phenolics were measured in the filtrate according to the method of TP he C.

Antioxidant Activity Assays
All antioxidant activity assays (including those using DPPH, ABTS, hydroxyl radicals, superoxide radicals, FRAP, CUPRAC, metal chelating, H 2 O 2 , β-carotene bleaching, and NO) were performed in accordance with our previous methods [51,52]; the specific experimental steps are as follows.
3.6.1. DPPH Assay Briefly, 100 µL of extract of PK insect gall in methanol and 100 µL of DPPH in methanol (50 µM) were mixed in a microplate and allowed to stand at room temperature for 20 min in the dark. The absorbance of the sample was recorded at 515 nm. Trolox was used as a positive reference, and its standard curve is y = 11.595x + 0.1821. The half-maximal inhibitory concentration (IC 50 ) values were calculated and expressed as the mean ± standard deviation (SD) in µg/mL.

ABTS Assay
Briefly, 190 µL of diluted ABTS solution and 10 µL of extract of PK insect gall in DMSO were mixed in a microplate and incubated for 20 min in the dark. The absorbance of the sample was recorded at 734 nm. Trolox was used as a positive reference, and its standard curve is y = 0.8026x + 11.878. The IC 50 values were calculated and expressed as the mean ± SD in µg/mL.

Hydroxyl Radical Assay
Briefly, 50 µL of extract of PK insect gall in DMSO, 50 µL of FeSO 4 solution (3 mM), and 50 µL of H 2 O 2 solution (3 mM) were mixed in a microplate and incubated for 10 min. After, 50 µL of salicylic acid solution (6 mM) was added and incubated at room temperature for 30 min in the dark. The absorbance of the sample was recorded at 492 nm. Trolox was used as a positive reference, and its standard curve is y = 0.0327x + 35.047. The IC 50 values were calculated and expressed as the mean ± SD in µg/mL.

Superoxide Radical Assay
Briefly, 45 µL of extract of PK insect gall in DMSO (10 mg/mL), 15 µL of p-nitroblue tetrazolium chloride (NBT) in DMSO (1 mg/mL), and 150 µL of NaOH in DMSO (50 µM) were mixed in a microplate, and the absorbance of the sample was recorded immediately at 560 nm against a blank sample (NBT was replaced with DMSO). Curcumin was used as a positive reference, and its standard curve is y = 84.38239x/122.02931 + x. The scavenging activity was expressed as % scavenging rate and was calculated as follows:

FRAP Assay
Briefly, 20 µL of extract of PK insect gall in DMSO and 180 µL of FRAP reagent were mixed in a microplate and incubated at 37 • C for 30 min in the dark. A calibration curve was produced based on FeSO 4 (0-600 mg/L) as a standard. The absorbance of the sample was recorded at 595 nm. The standard curve of ferrous ion is y = 4.416x + 0.087. Trolox was used as a positive reference. The FRAP was expressed as the Trolox Equivalent Antioxidant Capacity (TEAC FRAP ).

CUPRAC Assay
Briefly, 20 µL of CuCl 2 solution (100 mM), 50 µL of neocuproine in 96% ethanol (7.5 mM), 50 µL of NH 4 Ac solution, 20 µL of extract of PK insect gall in DMSO, and 30 µL of distilled water were mixed in a microplate and incubated at 50 • C for 20 min. This mixture was allowed to stand at room temperature for 10 min. The absorbance of the sample was recorded at 450 nm. Trolox was used as a positive reference, and its standard 3.6.12. β-Carotene Bleaching Assay Briefly, β-carotene solution was prepared by dissolving β-carotene (2 mg) in CHCl 3 (10 mL). Then, 2 mL of the solution was pipetted into a flask and vortex mixed with linoleic acid (40 mg) and Tween 40 (400 mg). After the removal of CHCl 3 , 100 mL of oxygenated ultra-pure water was added, and the emulsion was shaken vigorously. Aliquots (2.4 mL) of the emulsion were pipetted into different test tubes containing 0.1 mL of extract of PK insect gall in methanol (5 mg/mL). BHT and BHA were used as positive controls. In the control group, the extract of PK insect gall was replaced with water. When the sample was added to the emulsion, it was recorded as t = 0 min. The tubes were capped and placed in a water bath at 60 • C. The absorbance was recorded at 470 nm every 15 min until 120 min. The antioxidant activity coefficient (AAC) was calculated according to the following equation: where A A(120) is the absorbance of the antioxidant at 120 min, A C(120) is the absorbance of the control at 120 min, and A C(0) is the absorbance of the control at 0 min.

NO Assay
Briefly, 3 mL of extract of PK insect gall in methanol (1 mg/mL) and 3 mL of sodium nitroprusside solution (pH 7.4, 5 mM, in 0.1 M PBS) were mixed in an Eppendorf tube and incubated at 25 • C for 150 min. At intervals, 100 µL of the sample was pipetted from each Eppendorf tube onto a microplate containing 100 µL of Griess reagent. In the control group, the extract of PK insect gall was replaced with methanol. The absorbance was recorded at 546 nm against a blank sample (the Griess reagent was replaced with distilled water). Curcumin (0.1 mg/mL) was used as a positive reference.

Enzyme Inhibition Assays
All enzyme inhibition assays (including α-glucosidase, α-amylase, tyrosinase, urease, AChE, BChE, and xanthine oxidase) were performed in accordance with our previous methods [50]; the specific experimental steps are as follows.

α-Glucosidase Inhibition Assay
Briefly, 20 µL of extract of PK insect gall in DMSO (10 mg/mL) and 100 µL of yeast α-glucosidase (EC 3.2.1.20) solution (pH 6.9, 0.1 U/mL, in 0.1 M PBS) were mixed in a microplate and incubated at 25 • C for 10 min. Subsequently, 50 µL of p-nitrophenyl-α-D-glucopyranoside solution (pH 6.9, 5 mM, in 0.1 M PBS) was added and incubated at 25 • C for 5 min. Before and after incubation, the absorbance was recorded at 405 nm. In the control group, the extract of PK insect gall was replaced with DMSO. Acarbose was used as a positive reference. The inhibitory activity was expressed as % inhibition and was calculated as follows:

α-Amylase Inhibition Assay
Briefly, 20 µL of extract of PK insect gall in DMSO, 80 µL of distilled water, and 100 µL of porcine pancreatic α-amylase (EC 3.2.1.1) solution (pH 6.9, 4 U/mL, in 20 mM PBS) were mixed in an Eppendorf tube and incubated at 25 • C for 5 min. Subsequently, 200 µL of 0.5% starch solution (pH 6.9, in 20 mM PBS) was added and incubated at 25 • C for 3 min. Then, 200 µL of the mixture was removed from the Eppendorf tube and added to a separate Eppendorf tube containing 100 µL of 3,5-dinitrosalicylic acid reagent solution and placed in a 85 • C water bath. After 15 min, the mixture was removed from the water bath and diluted with 900 µL of distilled water. The absorbance was recorded at 540 nm. In the control group, the extract of PK insect gall was replaced with DMSO. In the blank group, the enzyme solution was replaced with PBS. Acarbose was used as a positive reference. The inhibitory activity was expressed as % inhibition and was calculated as follows: Before and after incubation, the absorbance was recorded at 405 nm. In the control group, the extract of PK insect gall was replaced with 10% DMSO. Donepezil was used as a positive reference. The inhibitory activity was expressed as % inhibition and was calculated as same as Section 3.7.1.

BChE Inhibition Assay
Briefly, 20 µL of extract of PK insect gall in 10% DMSO (1 mg/mL), 120 µL of PBS Before and after incubation, the absorbance was recorded at 405 nm. In the control group, the extract of PK insect gall was replaced with 10% DMSO. Donepezil was used as a positive reference. The inhibitory activity was expressed as % inhibition and was calculated as same as Section 3.7.1.

Tyrosinase Inhibition Assay
Briefly, 100 µL of L-tyrosine solution (pH 6.8, 5 mM, in 0.1 M PBS), 20 µL of PBS (pH 6.8, 0.1 M), and 40 µL of extract of PK insect gall in 10% DMSO (5 mg/mL) were mixed in a microplate. Subsequently, 40 µL of mushroom tyrosinase (EC 1.14.18.1) solution (pH 6.8, 200 U/mL, in 0.1 M PBS) was added and incubated at 37 • C for 20 min. The absorbance was recorded at 450 nm. In the control group, the extract of PK insect gall was replaced with 10% DMSO. In the blank group, the enzyme solution was replaced with PBS. Arbutin was used as a positive reference. The inhibitory activity was expressed as % inhibition and was calculated was calculated as same as Section 3.7.2.

Urease Inhibition Assay
Briefly, 60 µL of urea solution (pH 7.4, 100 mM, in 0.01 M PBS), 15 µL of jack bean urease (EC 3.5.1.5) solution (pH 7.4, 5 U/mL, in 0.01 M PBS), and 15 µL of extract of PK insect gall in PBS (pH 7.4, 0.01 M) were mixed in a microplate and incubated at 37 • C for 30 min. Subsequently, 60 µL of phenol reagents and 60 µL of alkali reagent were added and incubated at 37 • C for 30 min. The absorbance was recorded at 625 nm. In the control group, the extract of PK insect gall was replaced with PBS. In the blank group, the enzyme solution was replaced with PBS. Thiourea was used as a positive reference. The inhibitory activity was expressed as % inhibition and was calculated as same as Section 3.7.2. The absorbance was recorded at 290 nm. In the control group, the extract of PK insect gall was replaced with PBS. The enzyme solution was added to the microplate after adding HCl. In the blank group, the enzyme solution was replaced with PBS. Allopurinol was used as a positive reference. The inhibitory activity was expressed as % inhibition and was calculated as same as Section 3.7.2.

Stability of Methanol and Ethanol Extracts
The pH stability, thermal stability, and stability in a gastrointestinal tract model system of the extracts were determined in accordance with our previous methods [50]; the specific experimental steps are as follows.

pH Stability
The stability in acidic and basic environments was investigated using a methanol extract or ethanol extract of PK insect gall dissolved in deionized water with the pH adjusted to 1, 3, 5, 7, 9, or 11 using 1 M HCl or 1 M NaOH. The final concentration of methanol extract or ethanol extract was 50 mg/mL. After incubation at room temperature for 1 h, the pH of the mixture was adjusted to 7, and the TP he C and the ABTS scavenging abilities were examined.

Thermal Stability
To evaluate the thermal stability, methanol extract or ethanol extract of PK insect gall dissolved in deionized water (50 mg/mL, pH 7) was placed in test tubes with screw caps. The test tubes were placed in a boiling water bath (100 • C). Samples were removed after 0, 15, 30, 60, 120, 180, and 240 min and cooled in an ice water bath. The TP he C and the ABTS scavenging abilities were examined.

Modeling of the Stability in the Gastrointestinal Tract
First, 100 mL of methanol extract or ethanol extract of PK insect gall in distilled water (5 mg/mL) were mixed with 10 mL of PBS (pH 6.8, 10 mM) and incubated at 37 • C for 2 min (oral condition). Then, 0.5 mL of 1 M HCl-KCl buffer (pH 1.5) and 5 mL of pepsin solution (pH 1.5, 32 U/mL in 1 M HCl-KCl buffer) were added to the samples. The mixtures were incubated at 37 • C for 60 min (stomach condition). Thereafter, into the mixture was added 1 mL of 1 M NaHCO 3 together with 1 mL of mixture of bile and pancreatic juice (pH 8.2, 10 mg/mL of pancreatin, 14,600 U/mL of trypsin, 13.5 mg/mL of bile extract in 10 mM PBS), and the pH was adjusted to 6.8. The mixtures were incubated at 37 • C for 3 h (duodenal condition). The results were used for the determination of TP he C, and the ABTS scavenging abilities of methanol extract or ethanol extract during simulated gastrointestinal digestion were taken at 0, 0.5, and 1-4 h.

Oxidative Stability of Oils
The oxidative stability of EVOO and CPSO was evaluated according to our previous methods [50]; the specific experimental steps are as follows.
EVOO and CPSO were placed in separate flasks. Methanol extract or ethanol extract of PK insect gall was added to the EVOO and CPSO flasks at concentrations of 1000 and 250 µg/g. To compare with the stabilizing effect of methanol extract or ethanol extract, EVOO and CPSO were supplemented with synthetic antioxidants TBHQ and BHT at 200 µg/g. A control group was prepared without antioxidants. The flasks were left open and placed in an oil bath at 160 • C to simulate frying. Two samples from each category were removed from the flasks every 4 h for duplicate analysis. The oxidative stability of the oils was evaluated by measurement of the free acidity (percentage of oleic acid), peroxide values (milliequivalents of O 2 /kg oil), and ultraviolet absorption at 232 and 270 nm.

Cell Viability Assay
Cell viability was assayed using the TM 3 mouse cell line via the MTT assay, as previously described [52]; the specific experimental steps are as follows.
Briefly, 100 µL of a cell suspension (8 × 10 4 cells/mL) was seeded onto a microplate and incubated at 37 • C for 24 h in an atmosphere of CO 2 in a humidified incubator. After incubation, 100 µL of methanol extract or ethanol extract of PK insect gall was added. Five duplicate wells in each group were set up. Cell-free medium was employed as a blank group for zero adjustment during measurement. After incubation for 24 h and 48 h, respectively, 20 µL of MTT solution (5 mg/mL) was added and incubated at 37 • C for 4 h. The culture medium was removed, and 200 µL of DMSO was added to solubilize the formazan formed. The absorbance was recorded at 570 nm.

Statistical Analysis
One-way analysis of variance with the post hoc LSD test was conducted to determine significant differences in the total active constituents and biological activity assays between extracts (p < 0.05). Exploratory multivariate analysis was performed to cluster extracts, and Pearson's correlation analysis was completed to assess the relationships between the biological activities and the total active constituents.

Conclusions
The insect gall of PK has received little scientific attention; therefore, its chemical constituents, biological activities, and safety profile are unknown. This research fills some of these knowledge gaps. The outcomes of phytochemical analysis showed that the insect gall of PK contains 11 types of phytochemicals. the extracts of PK insect gall were prepared using seven solvents. The methanol and ethanol extracts had the highest antioxidant activity. The acetone extract exhibited high antigout and antihypoglycemic activity. The ethyl acetate extract was an excellent urease inhibitor. The dichloromethane extract exhibited inhibitory activity toward tyrosinase. In total, 25 specific compounds were identified from the methanol extract. The methanol and ethanol extracts exhibited excellent stability and safety. They could be used to stabilize olive and sunflower seed oils.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data presented in this study are available in the article.