Emerging Biopharmaceuticals from Pimpinella Genus

Evolved over eons to encode biological assays, plants-derived natural products are still the first dawn of drugs. Most researchers have focused on natural compounds derived from commonly used Pimpinella species, such as P. anisum, P. thellungiana, P. saxifrage, and P. brachycarpa, to investigate their antioxidant, antibacterial, and anti-inflammatory properties. Ethnopharmacological studies demonstrated that the genus Pimpinella has the homology characteristics of medicine and food and mainly in the therapy of gastrointestinal dysfunction, respiratory diseases, deworming, and diuresis. The natural product investigation of Pimpinella spp. revealed numerous natural products containing phenylpropanoids, terpenoids, flavonoids, coumarins, sterols, and organic acids. These natural products have the potential to provide future drugs against crucial diseases, such as cancer, hypertension, microbial and insectile infections, and severe inflammations. It is an upcoming field of research to probe a novel and pharmaceutically clinical value on compounds from the genus Pimpinella. In this review, we attempt to summarize the present knowledge on the traditional applications, phytochemistry, and pharmacology of more than twenty-five species of the genus Pimpinella.


Introduction
Secondary metabolites from nature, predominantly plant, are still elected as a first preference for drug discovery and serve as a hotpot because of their promising novel scaffolds against chronic diseases. Once the only thirst to cure diseases, elixirs and traditional medications allow for the more proficient approach to drug discovery. Plant-derived natural products were once the backbone of the pharmaceutical armamentarium, but the ready corresponding access to synthetic agents has discouraged the interest in maintaining a discovery paradigm from plants.
Recently, drug discovery from plants has sparked in many researchers and they have driven back their path toward terrestrial plants. Pimpinella is a species richness genus in the Umbelaceae family with unique morphological characteristics of monofoliate or compound leaves, three-out or one-to two-fold pinnate division. The flowers possessed characteristic umbels white or purplish-red, and ovoid and long ovoid was the most common fruit shapes [1]. The morphological characteristics of several major Pimpinella plants distributed in China are shown in Figure 1. By reviewing the literature, we discorvered about 150 species of Pimpinella were widely distributed in the area of Asia, Europe, and Africa, while only a few species were discovered in north and west of North America [2]. China, Turkey, and Iran were the three most abundant distribution centers of different species [3,4]. Approximately 39 species have been recorded in the Chinese Flora, most of which were

Folk-Medicine Application
The common ethnic uses of Pimpinella around the world are summarized as shown in Table 1, and it can be concluded that Pimpinella plants have the homology characteristics of medicine and food, wide varieties, and extensive traditional activities. In Asia, China was the country with the longest history and abundant resources in herbal remedy. P. diversifolia was used for the treatment of cold, indigestion, and diarrhea; P. candollean was eaten locally by Hmong as wild vegetables and used for resistance to stomach pain, bone pain, and rheumatism; P. thellungiana showed a remarkable anticoagulant effect [12]. Additionally, Koreans were keen to make P. brachycarpa delicious kimchi and were used medicinally for gastrointestinal dysfunction, asthma, and cough [13,14]. The seed of P. monoica native to India was used to fight stomachaches [15]. In the Middle East, species diversification of Pimpinellacould be observed in Turkey. The recorded endemic species, P. cappadocica [16], P. rhodantha [17], P. peregrine [18], and P. khorasanica [19] were applied in the therapy of deworming, digestion, sedation, expectoration, and increasing lactation. In Iranian folk studies, P. anisum seeds treated epilepsy since ancient times [20], while residents in Egypt and Lebanon attempted to use it to treat digestive and respiratory ailments [21,22]. Notably, Pimpinella's medicinal use is less prevalent in Europe and America, and the British and Brazilians usually used P. anisum as insect repellent, urinary disinfectant, and a deobstruent [23,24]. In Spain, France, and Italy, P. anisum was added to cooking, distilled alcoholic spirits, and confectionery industries as botanical spices [25,26].

P. anisum
Epilepsy Iran [20] P. anisum Insect repellents, stomach-cramping sedatives, diuretics, and urinary tract disinfectants England [23] P. anisum Used as plant spice to produce spirits drinks and confectionery Spain France [25] [25] Essential oil P. anisum Carminative, aromatic, disinfectant, and diuretic Iran [20] 3. Phytochemistry Recent investigations on chemical constituents of the genus Pimpinella identified 343 compounds, principally containing phenylpropanoids, terpenoids, flavonoids, coumarins, sterols, and organic acids. More than 80% of compounds were identified after 2000 ( Figure 2), among which phenylpropanoids, terpenoids, and flavonoids are the essential active components with the functions of an antioxidant, anti-inflammatory, and antitumor. Particularly, a unique and infrequent phenylpropanoid was found in the genus Pimpinella, named pseudo isoeugenol.

Phenylpropanoids
Phenylpropanoids are the main components occupying the dominant activity position in the volatile oils or extract of the Pimpinella genus, and a battery of studies have revealed anti-inflammatory, antibacterial, and antioxidant contributions [27]. Phenylpropanoids area class of natural compounds concatenated bya benzene ring and three straight-chain carbons (C 6 -C 3 units) with diverse activities. According to the skeletal characteristics of the phenylpropanoids in Pimpinella, they could be divided into three groups: pseudoisoeugenol, pseudoisoeugenol derivatives, and simple phenylpropanoids. Pseudoisoeugenol, as a representative chemical marker of Pimpinella, possessed a peculiar skeleton of 1-hydroxy-2propyl-4-methoxybenzene, which has been found exclusively in the genus Pimpinella so far [28]. Since the first pseudoisoeugenol was discovered from the P. anisum by G. T. Carter et al. [29] in 1977, scientists have successively obtained 12 pseudoisoeugenol (1-12) from the Chinese herb P. thellungiana, and llungianin A (1) and llungianin B (2), which presented significant antihypertensive activity [30][31][32][33][34][35][36][37].

Terpenoids
Terpenoids are a kind of active ingredient with diverse skeletons with manifold bioactivity and extensive distribution. The formula complies with the (C 5 H 8 ) n rule by polymerization of isoprene units in different linking ways. The long-term research by phytochemistshave certified that the volatile oil containing a large number of terpenoids has pervasively existed in Pimpinella, which released crucial activities of antibacterial, anti-inflammatory, and antidepressant activities. Notably, besides unique phenylpropanoids, the considerable quantity of specific C-12 sesquiterpene is another phytochemical marker distinguishing Pimpinella from other genera, such as geijerene-(107) and azulene-(109) type terpenes.
The volatile oil of P. anisum seed has attracted attention for its extensive biological activities such as antitumor, anti-inflammatory, and insecticidal agents. Numerous studies have characterized the ingredients in P. anisum, from which 46 monoterpenoids and sesquiterpenoids, including the principal ingredient linalool (101), were identified [6,[50][51][52][53]. Many researchers have analyzed the essential oil extracted from the foreign-sourced Pimpinella genus. A. V-Negueruela et al. [44] disposed of the aboveground parts of P. junoniae in Spain to obtain the oil, and α-zingiberene (20.6%) and α-pinene (17.9%) were the most abundant among 26 volatile constituents. N. Tabanca et al. [36,45] evaluated essential oils extracted from roots, stems, leaves, and fruits of four Pimpinella species (P. aurea, P. corymbosa, P. peregrine and P. puberula) on GC-MS, and a total of 95 terpenoids were identified. Meanwhile, further data comparison discovered that the main components of each plant in different parts possess differentiation, while only the oil from the root had a higher similarity, containing large quantities of epoxy pseudoisoeugenyl-2-methyl butyrate (26.8-42.8%). A series of Pimpinella plant's in vitro activity exploration, including the Turkish medicines P. anisetum [48], P. flabellifolia [48], and P. enguezekensis [51], the Iranian medicinal plants P. affinis [52] and P. khorasanica [19], the Indian herb P. monoica [15], and the Tunisian wild vegetable P. saxifrage [46], exhibited good antioxidant and antibacterial capacities, and GC-MS reports revealed terpenoids were dominant for their therapeutic effect.
Recently, researchers also conducted profound studies on the oil of domestic Pimpinella plants. X. W. Xu [53] and E. M. Suleimen et al. [54]

Terpenoids
Terpenoids are a kind of active ingredient with diverse skeletons with manifold bioactivity and extensive distribution. The formula complies with the (C5H8)n rule by polymerization of isoprene units in different linking ways. The long-term research by phytochemistshave certified that the volatile oil containing a large number of terpenoids has pervasively existed in Pimpinella, which released crucial activities of antibacterial, anti-inflammatory, and antidepressant activities. Notably, besides unique phenylpropanoids, the considerable quantity of specific C-12 sesquiterpene is another phytochem- In addition to terpenoids identified from volatile oils, S. Y. Lee's long-term research on chemical constituents of P. brachycarpa [13], two new sesquiterpenes (152)(153) and ten known terpenes (88-91, 128, 154-158) were isolated from the methanol extract of aerial parts. Ozbek et al. [16,17] obtained a new trinorguaian-type sesquiterpenoid (114) and a newly discovered triterpenoid glycoside (219) from P. cappadocica and P. rhodantha, respectively. Six triterpenoids (213-218) were isolated from P. anisum aqueous extract [55,56].

Flavonoids and Their Glycosides
Flavonoids are the focused topic of natural product excavation currently, and the majority of flavonoids in Pimpinella plants exhibited satisfactory antioxidant power in accordance with previous studies. Moreover, flavonoids showed more diversified bioactivities related to different functional groups, including phenolic hydroxyl, glycoside, and isopentyl. So far, more than 36 flavonoids have been isolated from this genus.

Flavonoids and Their Glycosides
Flavonoids are the focused topic of natural product excavation currently, and the majority of flavonoids in Pimpinella plants exhibited satisfactory antioxidant power in accordance with previous studies. Moreover, flavonoids showed more diversified bioactivities related to different functional groups, including phenolic hydroxyl, glycoside, and isopentyl. So far, more than 36 flavonoids have been isolated from this genus.

Organic Acids
Organic acids are widely distributed in leaves, roots, and fruits, and as aromatic plants, organic acid is a crucial element of volatile oil in the Pimpinella genus. Its structural types included aliphatic polycarboxylic acid, aromatic benzoic, and caffeic acid with

Pharmacology
Since the beginning of this century, due to the extensive use of the genus Pimpinella in traditional medicine, numerous scientific studies have demonstrated several ethnopharmacological properties from its extracts or compounds, including antibacterial, anti-inflammatory, insecticidal, antioxidant, and inhibitory enzyme activities [83]. In addition, some novel pharmacological activities such as antitumor, antidepressant, blood

Pharmacology
Since the beginning of this century, due to the extensive use of the genus Pimpinella in traditional medicine, numerous scientific studies have demonstrated several ethnopharmacological properties from its extracts or compounds, including antibacterial, anti-inflammatory, insecticidal, antioxidant, and inhibitory enzyme activities [83]. In addition, some novel pharmacological activities such as antitumor, antidepressant, blood pressure lowering, hypoglycemic, and liver protection have been gradually exploited recently. In our review, the effect of the Pimpinella species during the recent 8 years (2015-2022) was summarized, and specific pharmacological studies were discussed in the following paragraphs, as presumptively presented in Table 8 (Figure 9).

Antioxidant Activity
Plant-derived compounds have promising antioxidant activities (1-2). Within the past eight years, twenty studies have revealed the antioxidant properties in Pimpinella species, concentrating on P.anisum (60% of all studies). Seeds (70%) and aboveground parts (25%) were considered to be admirable candidates as antioxidants, and aromatics and flavonoids were identified as the dominant components. Experiments were divided into two categories: in vivo level and in vitro level. In vitro activity screening was a rapid and efficient antioxidant assay, with the precedence of animal studies constituting 90% of all tests.
Since 2015, only two in vivo tests were reported relating to the antioxidant activity of P. anisum. Favism is a metabolic disease of acute hemolytic anemia induced by bean consumption. In 2016, Kori, et al. demonstrated that pretreatment with P. anisum oil could block the oxidative stress effect of the causative agent to achieve a favism-protective effect by arresting the hydrolysis-of vicine ran convict to their aglylate free radical compounds (divicine and isouramil), and this effect related to anethole [7]. Ashtiyani's et al, study was aimed at exploring the alleviating effect of a P. anisum ethanol extract on gentamicin (GN)-induced Wistar rat model of nephrotoxicity by interfering with oxidative stress [84]. bThe results showed that P. anisum reversed the GN-induced increase in levels of plasma creatinine, BUN, MDA, and excretion of sodium and potassium and improved FRAP and GN-induced tubule damage.
On the other hand, the in vitro antioxidant performance of P. anisum was evaluated by utilizing different radical scavenging activities, such as DPPH and ABTS, reducing capacity assay (FRAP and PMCA), and β-carotene/linoleic acid determination. Many types of research showed satisfactory antioxidant properties of ethanol extract [84], aqueous extract [56], n-hexane extract [11], and volatile oil [50,85,86] of P. anisum seeds by various tests. As expected, further data comparison indicated that the DPPH clearance rate of oil exceeded 77% at the optimal concentration, superior to other types of extracts, and is recommended as a natural antioxidant. Furthermore, another analysis of oxidative-correlative components revealed that P. anisum oil possessed a positive correlation with the total amount of phenols and polysaccharides [9] and a negative correlation with the total amount of sterols [78].

Antioxidant Activity
Plant-derived compounds have promising antioxidant activities (1-2). Within the past eight years, twenty studies have revealed the antioxidant properties in Pimpinella species, concentrating on P. anisum (60% of all studies). Seeds (70%) and aboveground parts (25%) were considered to be admirable candidates as antioxidants, and aromatics and flavonoids were identified as the dominant components. Experiments were divided into two categories: in vivo level and in vitro level. In vitro activity screening was a rapid and efficient antioxidant assay, with the precedence of animal studies constituting 90% of all tests.
Since 2015, only two in vivo tests were reported relating to the antioxidant activity of P. anisum. Favism is a metabolic disease of acute hemolytic anemia induced by bean consumption. In 2016, Kori, et al. demonstrated that pretreatment with P. anisum oil could block the oxidative stress effect of the causative agent to achieve a favism-protective effect by arresting the hydrolysis-of vicine ran convict to their aglylate free radical compounds (divicine and isouramil), and this effect related to anethole [7]. Ashtiyani's et al, study was aimed at exploring the alleviating effect of a P. anisum ethanol extract on gentamicin (GN)-induced Wistar rat model of nephrotoxicity by interfering with oxidative stress [84]. bThe results showed that P. anisum reversed the GN-induced increase in levels of plasma creatinine, BUN, MDA, and excretion of sodium and potassium and improved FRAP and GN-induced tubule damage.
On the other hand, the in vitro antioxidant performance of P. anisum was evaluated by utilizing different radical scavenging activities, such as DPPH and ABTS, reducing capacity assay (FRAP and PMCA), and β-carotene/linoleic acid determination. Many types of research showed satisfactory antioxidant properties of ethanol extract [84], aqueous extract [56], n-hexane extract [11], and volatile oil [50,85,86] of P. anisum seeds by various tests. As expected, further data comparison indicated that the DPPH clearance rate of oil exceeded 77% at the optimal concentration, superior to other types of extracts, and is recommended as a natural antioxidant. Furthermore, another analysis of oxidativecorrelative components revealed that P. anisum oil possessed a positive correlation with the total amount of phenols and polysaccharides [9] and a negative correlation with the total amount of sterols [78].
Meanwhile, many studies clarified the antioxidant effect of different Pimpinella species abroad, providing a logical basis for the rational choice of the Pimpinella plant. Ozbek et al. also proved the superior antioxidant activities of P. cappadocica [16] and P. rhodantha [17], which were consistent with the flavonoid glycosides content, while the antioxidant capacities of P. enguezekensi [51] and P. anthriscoides [65], newly discovered species in eastern Anatolia, that were attributed to high trans-anethole concentration. The antioxidant characterizations of ethyl acetate extracts from Indonesian P. alpine [88] and Iranian P. affinis [89] were conducted by in vitro screening with IC 50 values of 53.07 and 74.90 µg/mL, respectively. A study in 2019 [46] discovered that 3% of P. saxifraga oil exhibited significant antioxidant activity and DNA protection potential, correlating with the proportion of phenolic compounds [90], which indicated it could be used as a new natural antioxidant candidate added to the sodium alginate coating in the preservation of cheese.

Antibacterial Activity
Bacterial infection is the main cause of morbidity and mortality throughout the world. Since antiquity, scientists have been interested in its bacteriostatic potential due to the characteristic volatile compositions in the Pimpinella species. In Table 8, most data concerning P. anisum oil presented that phenyl propanes, especially anethole and its isomers, were the predominant components accounting for 98% of its content [149]. Various test procedures were conducted, such as disk diffusion, agar diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) using in vitro conditions to explore the antagonistic activity of microorganisms of extracts from different species.
Since 2015, 19 reports have multidimensionally characterized the antimicrobial activity of P. anisum, accounting for 79%. The essential oil from P. anisum has been triumphantly developed as a target preparation, and with advances in biological materials, the combination of PLA film materials, nano emulsions, and gel materials with oil has been affirmed as a new dosage form, which could greatly improve its antibacterial ability. In terms of antibacterial experiments, many studies demonstrated that oil and polysaccharide from P. anisum seeds and fruits exhibit antibacterial activity against a battery of gram-negative and gram-positive bacterium (Table 8) [8,9,91,92,150]. Noteworthily, fire blight was a devastating disease of commercial crops of Rosaceae, ascribing to the highly infectious bacteria Erwiniaamylovora, and Akhlaghi et al. found that P. anisum oil showed above-average antibiotic ability with a MIC of 31.25 µg/mL [93].
In antifungal experiments, P. anisum oil-hydrogel formulation was successfully prepared by the freeze-drying method, which was suitable for vaginal delivery systems and showed restraining activity against Candida albicans, C. glabrata, and C. parapsilosis [94]. Currently, aromatic plants have attracted interest for scientists as sources of natural antimicrobials due to the increased resistance of pathogenic fungi. Khosravi et al., confirmed P. anisum oil was sensitive to Fusarium solani emerging from patients with onychomycosis with a MIC ranging from 50 to 490 µg/mL [95].
In another dermatophyte infections study [96], combined treatment with terbinafine and P. anisum oil showed that oil enhanced the activity of terbinafine against Trichophytonrubrum and T. mentagrophytes with a 4-fold reduction in the MIC. The combination therapy had a synergistic effect on reducing the concentration of antifungal drugs and the appearance of resistant strains than monotherapy. A. J. Obaid et reported that P. anisum oil down-regulated the keratinase gene expression of T. rubrum by 0.079 compared with control (1.00), conducing to target determination during drug development [97].
In recent years, with the continuous improvement of consumer requirements for food safety, the application of P. anisum oil as a natural antibacterial agent has been greatly promoted in the food domain. Many microbiology experiments [98,99] demonstrated that P. anisum oil exerted an inhibitory effect on the growth of the food-born germ Clostridium perfringens and several mycete by controlling of mycelium growth and spore germination [100]. Khoury et al. further integrated with qRT-PCR to reveal the modulation of 5 µL/mL P. anisum oil on the ochratoxin A production during grape brewing by downregulating the expression of Aspergillus carbonarius biosynthesis-related genes (acOTApks, acOTAnrps, acpks gene) and growth-regulating genes (laeA and vea gene) [101]. Noori et al., research in 2021 showed a concentration-dependent inhibitory effect on Listeria monocytogenes and Vibrio parahaemolyticus by adding P. anisum oil to a novel polylactic acid (PLA)-based composite film for food packaging [102]. Ultrasound-assisted P. anisum oil-based nanoemulsion prevented microbial contamination induced by 5 bacteria and 14 food-contaminating fungi compared with pure extract and is recommended as a green food antiseptic [87,[105][106][107].
In addition to P. anisum, many studies exhibited the antimicrobial potentials of different Pimpinella species from around the world, including, P. alpine [88], P. saxifrage [46], P. enguezekensis [51], P. affinis [52], and P. anthriscoides [65], which showed the moderate bacteriostatic effect against a battery of microorganisms, suggesting development as an alternative for P. anisum.

Anti-Inflammatory Activity
The cause of body inflammation is either infection or physical/chemical damage. In that case, blood starts oozing out into tissues from blood vessels (5)(6). P. anisum has been approved by the Committee of Herbal Products of the European Medicines Agency (EMA) for a therapeutic schedule of mild indigestion and an expectorant for coughs due to its traditional effects on respiratory disorders. As the literature ascertained, the genus Pimpinella exerted an anti-inflammatory effect by regulating the expression of proinflammatory cytokines (IL-1, IL-8, and TNF-α), and anethole (40) from the volatile oil was the prime ingredient [96]. However, there was little research on its anti-inflammatory mechanism in respiratory tissues. T. P. Domiciano et al. previously provided preclinical evidence that anethole (40) inhibited the production or release of PGE 2 and NO in acute inflammation in animals [106]. R. Iannarelli et al. [107] further revealed that P. anisum oil acted as a remarkable anti-inflammatory by reducing the expression of IL-1 and IL-8 in LPS-induced tracheal epithelial HBEpC and HTEpC lines and promoting the secretion of Muc5ac. Another study on the respiratory system examined the effects of anethole (40) on the inflammatory status of lung and liver cells after exposure to airborne pollution of particulate matter (PM). In PM 2.5 -induced BEAS-2B and HepG2 cells, anethole (40) reduced the levels of IL-6 and IL-8 by 96% and 87%, respectively, demonstrating it is a natural therapeutic agent to counteract PM-induced inflammation [108]. Recently, based on analysis of ovalbumin (OVA)-induced allergic rhinitis (AR) model mice, C. S. Liao's team found that the anti-inflammatory response of BLAB tea containing P. anisum was relevant to the suppression activity on the accumulation of inflammatory cells and the release of Th2 and histamine in the nasal mucosa, NALF, and serum, and induction of the production of Th1 and Treg [109]. Another P. anisum study [9] indicated that polysaccharide extract mediated anti-inflammatory effects by improving edema and reducing MDA and SOD levels of oxidative stress indexes in muscle in carrageenan-induced foot swelling in mice.

Anti-Tumor Activity
It is important to note that the antitumor activities of genus Pimpinella have been verified at the cellular level and in animal studies, while few studies report on clinical applications. Terpenoids from P. anisum seed are the dominant antitumor compounds. A 2015 study showed that treating HepG2 cells with P. anisum oil for 24 h caused concentrationdependent and significant cytotoxicity [110]. Nowadays, silver nanoparticles (AgNPs) provide a new pathway for the utilization of natural products and the importance of drug release. Alsalhi et al., designed a green synthetic route in 2016 to prepare AgNPs containing a P. anisum aqueous extract, which exerted obvious antitumor effects on human neonatal skin stromal cells and colon cancer cells [111]. S. Devanesan et al. conducted an in-depth study on the pharmacological mechanism of AgNPs in the colorectal cancer cell (CRC) [112]. Interestingly, synthetic AgNP could selectively destroy CRC via the inhibition of proliferation, arresting the cell cycle at the G2/M phase, and inducing apoptosis, indicating that composite nanomedicines may pioneer new approaches for prospective anticancer therapy. A. Mahmoud et al. reported a novel sesquiterpene lactone pimpinelol (205) from P. haussknechtii and demonstrated reduction viability against human breast cancer cell line (MCF-7 cells, IC 50 : 1.06 µM) by inducing protein aggregation and endoplasmic reticulum stress at the cytokine levels [10].

Hypoglycemic Activity
Diabetes is a lifelong metabolic disease characterized by hyperglycemia, leading to a variety of deadly complications. Previous reports have confirmed that the ethanol extract of P. brachycarpa possesses the capacity for precaution of hyperglycemia and remission of oxidative stress in type II diabetic mice [14]. Since 2015, studies paid attention to P. anisum in controlling hyperglycemia and preventing diabetes complications. Preliminarily, M. Bonesi et al. evaluated the inhibitory activity of P. anisum seed on two key enzymes associated with type II diabetes, and it exhibited moderate inhibition against α-amylase and α-glycosidase with IC 50 values of 692.6 ± 5.2 and 73.9 ± 2.2 µg/mL, respectively [11]. Secondly, a 2020 study using a streptozotocin (STZ)-induced diabetic rat model observed that β-cell structure was significantly improved, insulin immune response was enhanced, and pancreatic acinus and amylase levels were reduced in the P. anisum-treated group compared to diabetic-control. The authors attributed the beneficial effects of P. anisum extract to its hypoglycemic and antioxidant properties, as oxidative stress plays a critical role in the development and progression of diabetes. In this study, the P. anisum-treated group significantly reduced SOD and CAT and increased their levels of lipid peroxidation marker MDA, which plays a role in lowering blood glucose. In addition, in immunohistochemical experiments, it could be observed that compared with diabetic control groups, the caspase 3 immunoreaction (22.34 ± 1.27 vs. 52.96 ± 2.32) and beclin 1 immunoreaction (31.55 ± 1.05 vs. 46.85 ± 1.30) were significantly decreased in the P. anisum-treated group (p < 0.001). These results indicated that P. anisum could significantly down-regulate the autophagy regulation marker beclin 1 and apoptosis marker caspase 3 in the pancreas, also relating to its antioxidant properties. Finally, M. Hashemnia et al. explored the potential of P. anisum on skin ulceration complications induced by diabetes from a new perspective of wound healing [114]. P. anisum reversed oxidation changes of MDA and GSH in wound skin (p < 0.05) and significantly reduced the wound size and the number of inflammatory cells while enhancing the re-epithelialization rate, collagen content, and fibroblast reaction, promoting festering wound reparation in diabetic rats.

Hypotensive Activity
A report in 2017 demonstrated that the ethyl acetate and ethanol extract of P. brachycarpa has a significant antihypertensive function in hypertensive model rats [115]. Further invitro exploration revealed it exhibited a dose-dependent inhibitory effect on an angiotensin-converting enzyme in the range of 0.5-10 mg/mL, and 80% ethanol extract presented the highest inhibitory rate. However, the effective ingredients and mechanism of hepatoprotective activity need to be further clarified. Another study in 2019 confirmed that an aqueous extract of P. anisum seed had the beneficial effect of lowering arterial blood pressure in rats and further explored its mechanism by estimation of different models [116]. V.B.C. Pontes et al. successively eliminated the actions of diuresis, angiotensin receptor antagonism, and β-receptor blockade of P. anisum. Additionally, it proved to act as a calcium channel antagonist to act as a hypotensive agent by inhibiting Ca 2+ influx.

Insecticidal Activity
Since the 20th century, the wide application of pesticides has led to the rapid development of agriculture and a booming increase in output. However, the increasing pests' resistance and soaring pollution in the environment and food caused by synthetic pesticides have motivated researchers to explore natural botanicals as sources of new insecticides, such as Pimpinella essential oils.
A 2018 study [117] used P. anisum oil to explore the toxicity of agricultural pests and the safety of beneficial insects, and the results displayed noteworthy insecticidal effects against two pathogenic insects, Culex. quinquefasciatus (LC 50 = 25.4 µL/L) and Scaphoideus littoralis (LD 50 = 57.3 µg/L). Nevertheless, it was not toxic to beneficial insects in comparison with α-cypermethrin at the same lethal concentration. Similar inferences were drawn from another nine studies by contact and fumigation tests [118][119][120][121][122][123][124][125][126]. A.Hatege kimana et al. revealed another pathway in the eradication of the pest (Acanthoscelidesobtectus) by reducing fecundity (egg production) and fertility (egg hatch ability/progeny production) [127]. Ulteriorly, in vitro tests observed activity decline of AchE in two-spotted spider mites after P. anisum management, which was attributed to the high-content ingredients, such as Eanethole, isoeugenol, and α-pinene [86]. In addition, green insecticides with the cooperation of emerging eco-friendly substances and natural ingredients are perceived as a strategy. K. A. Draz et al. prepared P. anisum oil of nanoemulsions (2500 mg/L) to eliminate the emergence of Sitophilus oryzae and Triboliumcastaneum by 94.6% and 84.5%, respectively, which exceeded the values compared to that of pure oil; it had no adverse impact on the germination rate of wheat [128]. Concurrently, various attempts [129][130][131][132][133] have proved P. anisum-nanoformulation possessed considerable repellent and toxic activities against Bactroceraoleae and other crop pests.
In addition to the management of crop pests, P. anisum oil had an excellent performance on larval elimination and cutting-off transmission against pests spreading epidemic diseases. Numerous studies have provided convictive evidence of larval killing and oviposition deterrent activities of P. anisum on pestiferous pests, the vector of dengue, human African trypanosomiasis, and filariasis [48,[136][137][138][139][140]. A. T. Showler et al. further demonstrated p-anisaldehyde was a botanical ingredient inhibiting the reproduction of pests [139,140]. To develop efficient mosquitocide, S. S-Gomez et al. encapsulated P. anisum oil in nanoparticles loaded with zein to overcome the defects of high degradability and low persistence and successfully applied it to mosquito larvicide [141]. Overall, Pimpinella oil not only combated insect vectors but also prevented crops and other organisms from toxic damage, representing a milestone in the commercial development of green-based insecticide formulations.

Enzymes Inhibitory Activity
O. H. Chan's research declared that the ethanol extract of P.brachy carp regulated the enzymes CYP1A2, 2B6, and 3A4 by concerted inhibition, while it affected the enzymes CYP2C19 and 2D6 by competitive inhibition [142]. Furthermore, G. Zengin et al. evaluated the enzymatic inhibition of Turkish P. anthriscoides on tyrosinase, α-amylase, α-glucosidase, AChE, and BChE by invitro tests [65]. Since 2015, six studies focused on exploring the P. anisum-derived enzyme inhibitor. On one hand, scientists actively probed plant extracts, and a sol-gel GSTA1-1macroarray high-throughput screening tool was independently developed for celerity determination of the GST-inhibitory activity of P. anisum (IC 50 = 3.40 ± 0.83 µg/mL) [143]. Gout was induced by excessive accumulation of uric acid due to xanthine oxidase (XO), which has the function of oxidizing hypoxanthine to xanthine and uric acid in an overactive state. L. Bou-Salah et al. revealed that P. anisum oil inhibited the activity of human-original XO (IC 50 = 2.37 ± 0.23 µg/mL), discussing new tactics for gout treatment [144]. RALDHs were assigned to convert retinaldehyde to retinoic acid (RA), acting as the dominant mechanism in RA signaling pathways and relevant cancers. The current study indicated that ethanol extracts of P. anisum selectively and intensively inhibited RALDH3 expression, while it did not modulate RALDH1 and RALDH2, highlighting the selectivity of that in the regulation of RALDHs and the RAgoverned metabolic process [145]. On the other hand, studies on P. anisum ingredients found that abundant bergapten (256), iso-pimpinellin (274), and methoxsalen (275) were inhibitors of CYP-1A2, which are involved in drug metabolism and carcinogenic bioactivity [70]. However, phenolic ingredients exerted remarkable inhibition against AChE and BChE with IC 50 values of 0.07 and 0.34 µg/mL, respectively [56,66].

Antidepressant Activity
Depression and anxiety disorders are commonly believed to be stress-related mood disorders, invariably accompanied by various diseases and premature aging in severe cases. Many studies have manifested that the antidepressant effect of genus Pimpinella extracts is associated with neurotransmitters, genetic polymorphisms, endocrine system abnormalities, and cytokine levels [151]. A reversion of anxiety and depression and amelioration of memory formation in model mice by total extract of P. anisum [152] (100 mg/kg) and volatile oil of P. peregrine [18] could be observed according to researchers. However, precise elucidation of the mechanism was needed. Therefore, the in-depth study focusing on P. anisum oil by Koriem et al. [146] found that levels of 5-HT, DA, NE, GABA, and IL-10 were significantly reduced (p < 0.001) and the levels of inflammatory cytokines IL-1β, IL-6, TNF-α, and Ki-67 were significantly increased (p < 0.001) after oral administration of P. anisum oil compared with chronic mild stress (CMS) model rats, bringing the cerebral cortical and hippocampal levels close to normal. As is known, the inflammatory factors mainly occurred in allergy conditions. TNF-α represented an inflammatory factor in neurons where IL-1β produced inflammation through monocytes and macrophages; IL-6 and IL-10 had a vital role in the neuronal response to injury, while Ki-67 represented a nuclear protein, which was associated with cellular multiplication. These results confirmed the efficacy of P. anisum oil in the treatment of depression by inhibiting the inflammation of the cerebral cortex and hippocampus. It is worth noting that El-Shamy et al. concluded with conflicting results compared to Koriem et al., using the same animal models and experimental procedures as they attributed the depression-improving effect to its antioxidant activity [147]. The reason was decreased levels of GSH-Px, GST, GSH, and CAT, while increased levels of MDA and NO were observed in the cerebral cortex and hippocampus. Additionally, M-jahromi et al. [148] selected 120 patients with depression suffering from irritable bowel syndrome (IBS) to provide clinical evidence of the antidepressant effect of P. anisum. The P. anisum-treated group preferentially alleviated mild or moderate depressive symptoms in IBS patients compared to control and placebo groups making it a prospective and economical option for depressive patients.

Other Activities
In addition to the above pharmacological activities related to traditional usage, many novel pharmacological properties have been excavated from the genus Pimpinella. For example, the ethanol extracts of P. anisum combated uterine contractions by inhibiting L-type Ca 2+ channels and blocking Ca 2+ influx [153], and the polysaccharide extract accelerated wound healing [9]. Mosavata and his team implemented placebo-controlled trials to demonstrate that P. anisum ameliorated the distress of migraine [154] and premenstrual syndrome [155]. Moreover, umbelliprenin (276) in P. anisum has been proven to be a potential skin-whitening agent [71]. These data were anticipant of genus Pimpinella for drug exploitation in the treatment of various diseases.

Conclusions and Perspective
In this review, the traditional uses, chemical constituents, and modern pharmacological activities of the genus Pimpinella were summarized. Conclusively, genus Pimpinella principally contained phenyl propanoids, terpenoids, flavonoids, coumarins, sterols, and organic acids with a broad spectrum of biological activities, such as antioxidant, antibacterial, anti-inflammatory, antitumor, hypoglycemic, hypotensive, insecticidal, inhibitory enzyme, and antidepressant activities. Some Pimpinella cultivars could be applied as natural sources of edible vegetables, and essential oil was the important raw material for the production of green insecticides and condiments of alcoholic beverages.
This review is prepared to provide an overview of the knowledge of the last eight years (from 2015 to 2022) and to make suggestions for filling the gaps available in the literature for this genus. However, there were still some shortcomings during the overview of the genus Pimpinella, and suggestions were made for filling the gaps available in the literature for this genus. The mechanism, target, toxicity, and clinical application of the pharmacology needed to be further studied and discussed. Firstly, the species Pimpinella were abundant with similar appearance in China, and most were used as folk medicine. Detailed identification and quality standard were conducted only in P. anisum and P. thellungiana. Hence, it is urgent to establish a complete quality standard for Pimpinella plants to prevent the mixeduse phenomenon. Secondly, despite the increasing demand for pharmacological research on the genus Pimpinella recently, such as antioxidant, anti-inflammatory, antitumor, antidepressant, and hypoglycemic effects, more attention should be paid to the relevant clinical research. The therapeutic properties recorded in medical books of various countries of all ages should be appreciated. For example, the traditional curative effect of P. anisum in the gastrointestinal tract and digestive function documented in many places has not been confirmed by uniting with modern scientific methods, which provides new directions for the future. Finally, P. anisum's essential oil, aqueous, or organic solvent extracts are often applied for pharmacological investigation. To better clarify the pharmacological activity of P. anisum, the bioactivity-oriented separation method can be adapted to excavate the bioactive components and maximize utilization.