Journal of Functional Foods 75 (2020) 104213

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Journal of Functional Foods

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Limonoids from Citrus: Chemistry, anti-tumor potential, and other T bioactivities Yu-Sheng Shia,b,1, Yan Zhangc,1, Hao-Tian Lia, Chuan-Hai Wud, Hesham R. El-Seedie,f, ⁎ ⁎ Wen-Kang Yed,g, Zi-Wei Wangd, Chun-Bin Lia, Xu-Fu Zhangc, , Guo-Yin Kaib, a Key Laboratory of Biotechnology and Bioresources Utilization, Educational of Minister, College of Life Science, Dalian Nationalities University, Dalian 116600, China b Laboratory of Medicinal Plant Biotechnology, College of Pharmacy, Zhejiang Chinese Medical University, Hangzhou 311402, China c School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China d Department of Ocean Science, Division of Life Science and Hong Kong Branch of the Southern Marine Science and Engineering Guangdong Laboratory, The Hong Kong University of Science and Technology, Hong Kong, China e Pharmacognosy Group, Department of Medicinal Chemistry, Uppsala University, Biomedical Centre, Uppsala 75 123, Sweden f International Research Center for Food Nutrition and Safety, Jiangsu University, Zhenjiang, 212013, China g SZU-HKUST Joint Ph.D. Program in Marine Environmental Science, Shenzhen University, Shenzhen 518061, China

ARTICLE INFO ABSTRACT

Keywords: Citrus limonoids are tetranortriterpenoids compounds mainly found in oranges, lemons, grapefruits, and other Rutaceae fruits of Citrus. They are proved to be the leading cause of bitterness in Citrus fruits and are mainly consumed for Citrus therapeutic purposes and as food. Numerous studies have focused on Citrus limonoids and intend to develop new Limonoid chemotherapeutic or complementary medicine in recent years. Citrus limonoids showed various bioactivities Chemical structure such as anti-tumor, antioxidative, anti-inflammatory, anti-neurological diseases, immunomodulatory, anti-in- Bioactivity sect, anti-bacteria, antiviral activities, etc. This review summarized limonoids from Citrus to date, along with Anti-tumor their chemical structures and biological activities with a particular focus on their anti-tumor potential.

1. Introduction were shown to be well tolerated by non-cancerous cell lines (Gualdani, Cavalluzzi, Lentini, & Habtemariam, 2016; Jayaprakasha et al., 2008; Limonoids, also known as tetranortriterpenoids, exist extensively in Jayaprakasha, Jadegoud, Gowda, & Patil, 2010), animal models Rutaceae and Meliaceae and less frequently in the Cneoraceae and (Guthrie, Morley, Hasegawa, Manners, & Vandenberg, 2000; Miller, Simaroubaceae (Tundis, Loizzo, & Menichini, 2014). The studies of li- Porter, Binnie, Guo, & Hasegawa, 2004; Tanaka et al., 2000, 2001), and monoids originate from the attempt on investigating responsible che- human body (Kelley et al., 2015; Manners, Jacob, Breksa, Schoch, & micals for bitterness in Citrus fruit, and the name of limonoid stems Hasegawa, 2003). Based on the above properties and bioactivities, li- from limonin, the first bitter tetranortriterpenoid separated from Citrus monoids present potential applications in food and pharmaceutical in- (Sandjo & Kuete, 2013). Limonoid aglycones, some of which contribute dustries (Patil, Jayaprakasha, Murthy, & Vikram, 2009; Roy & Saraf, to the bitter taste of Citrus fruits, and their glycosides widely occur in 2006), and have been used as functional food additives, pesticides, and the seeds and fruits of Citrus (Matheyambath, Padmanabhan, & feed additives. Paliyath, 2016). Limonoid from Citrus has been a research interest due This review covers the natural limonoids from Citrus to date and to its contribution to the bitter taste of Citrus fruits, which harms the their occurrence in different species, along with their chemical struc- global Citrus fruit and juice industry. In recent decades, limonoids from tures and biological activities. The multiple pathways that limonoids Citrus is becoming a research hotspot owning to their diverse bioac- were shown to be involved in were summarized, and the pharmacolo- tivities in vitro and in vivo such as anti-tumor, antioxidative, anti-in- gical potential of limonoids in different diseases was discussed to fa- flammatory, anti-neurogenic diseases, immunomodulatory, in- cilitate in-depth researches regarding bioactivity and the application of secticidal, antibacterial, and antiviral activities (Zhang et al., 2013). limonoids. Furthermore, limonoids from Citrus, as food-derived phytochemicals,

⁎ Corresponding authors. E-mail addresses: [email protected] (Y.-S. Shi), [email protected] (X.-F. Zhang), [email protected] (G.-Y. Kai). 1 Equal contribution. https://doi.org/10.1016/j.jff.2020.104213 Received 27 May 2020; Received in revised form 12 September 2020; Accepted 15 September 2020 Available online 23 September 2020 1756-4646/ © 2020 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Y.-S. Shi, et al. Journal of Functional Foods 75 (2020) 104213

Dragull, & Wong, 2008), deoxylimonol (27)(Bennett & Hasegawa, 1982) were the most representative limonoids. The cleavage of lactone bond in ring D of limonin generated limonoic acid A-ring lactone (29) (Maier & Margileth, 1969) and the further oxidation product 17-de- hydrolimonoate A-ring lactone (30)(Herman, Fong, & Hasegawa, 1992). Limonin glucoside (72)(Hasegawa, Bennett, Herman, Fong, & Ou, 1989) was the glycosylation product of limonin. The structural features of nomilin-type were similar to limonin-type, except for the ring A, which was a seven-membered lactone with the oxygen atom positioned between C-3 and C-4. The main nomilin-type limonoids included nomilin (4)(Ishii, Ohta, Nogata, Yano, & Hasegawa, 2003), obacunone (1)(Kim, Jayaprakasha, & Patil, 2014), 7α-obacunol (2)(Bennett & Hasegawa, 1982), 7α-obacunyl acetate (3)(Bennett & Hasegawa, 1982), obacunone 17-β-D-glucoside (65)(Hasegawa et al., 1989), deacetylnomilin (5)(Hamdan et al., 2011), 6-keto-7β-deace- tylnomilol (6)(Bennett & Hasegawa, 1981), 6-keto-7β-nomilol (7) (Herman, Bennett, Ou, Fong, & Hasegawa, 1987), citrusin (46)(Chan, Li, Shen, & Wu, 2010) and nomilin glycoside (66)(Hasegawa et al., 1989). Nomilinic acid type differs from nomilin type in the structure ofring Fig. 1. Structural features of limonin, the most representative limonoid from A. The lactone bond in ring A is cleaved and culminated in a C-3 car- Citrus. boxyl or a methoxycarbonyl group in nomilinic acid-type limonoids. Nomilinic acid (8)(Hashinaga, Fong, & Hasegawa, 1990), obacunoic 2. Chemistry acid (14)(Bennett, Hasegawa, & Herman, 1989), methylnomilinate (9) (Bennett & Hasegawa, 1980), methyldeacetyl nomilinate (11)(Bennett Limonoids are highly oxidized triterpenoid displaying homogenous & Hasegawa, 1981), calamin (12)(Bennett & Hasegawa, 1981), nomi- stereochemistry, featured with a structure either containing or origi- linic acid glucoside (8)(Hashinaga et al., 1990) and deacetylnomilinic nated from a precursor with a 4,4,8-trimethyl-17-furanylsteroid scaf- acid 17-β-D-glucoside (67)(Bennett et al., 1989) are the typical limo- fold. Citrus limonoids were characterized with a furan ring linked to the noids of this type. Overall, the diversification of Citrus limonoids mostly D-ring at C-17, and oxygen-containing functional groups at C-3, C-4, C- occurs in A and B rings along with different oxidized level, while D ring 7, C-16 and C- 17 (Fig. 1). The structural diversification of limonoids is either opened or lactonized. from Rutaceae is less than that in Meliaceae and are mostly limited to the change in A and B rings. The biosynthesis pathway of limonoids remained largely unin- 3. Distribution vestigated and was only tentatively proposed (Wang et al., 2017). Li- monoids were commonly considered to derive from euphane (20-S) or Limonoids are widely distributed in many Citrus fruits such as sweet tirucallane (20-R) involved in the triterpenoid biogenetic route (Fig. 2). orange (Citrus sinensis), lemon (Citrus limon), grapefruit (Citrus paradisi), The Δ7-bond was oxidized into 7-epoxide, which was open subse- lime (Citrus aurantiifolia), sour orange (Citrus aurantium), pummelo quently. Then, Me-14 transferred to C-8 through the Wagner-Meerwein (Citrus maxima)(Russo et al., 2016). The detailed distribution of li- rearrangement, accompanied by the formation of the 7-OH and a monoids in different Citrus species so far is summarized in Table 1. double bond at C-14/C-15. After that, the side chain cyclized and lost Generally, the water-insoluble limonoid aglycones such as limonin, four carbons to form the 17 β-furan ring. Based on the basic skeleton of nomilin, obacunone, ichangin, deacetyl nomilin are mainly distributed limonoids, a series of oxidations and skeletal rearrangements occurred within seeds and peels. In contrast, the water-soluble limonoid gluco- to generate a variety of limonoids. For example, rings A and ring D are sides, such as limonin glucoside, nomilin glucoside, obacunone gluco- sometimes oxidized A and D-seco limonoids by Baeyer-Villiger oxida- side, and nomilinic acid glucoside, are more abundant within juices and tion reaction of the C-3 and C-16 keto groups; rearrangement of the ring pulps (Breksa, Hidalgo, & Yuen, 2009). Several recent reports have A result in the maturation of typical limonoids such as limonin. The described the limonoid content in different parts of Citrus fruits (Celano biosynthesis correlation between limonoids from Citrus has been sys- et al., 2019; Russo et al., 2016; Wang et al., 2016). As an example, the tematically reviewed (Hasegawa, 2000). Recently, the genetic founda- concentrations of limonoid content were 90.8, 514.2, 804.8 and tion of limonoid biosynthesis was investigated (Wang et al., 2017), 6828.1 mg/kg for the juice, pulp, peel, and seed of Citrus bergamia fruit, CiOSC was shown to be the gene encoding the critical enzyme oxi- respectively (Table 2)(Russo et al., 2016). According to those data, a dosqualene cyclase (OSC) in the limonoid biosynthesis, and cytochrome Citrus fruit could provide an intake of limonoids in dozens of milli- P450s (CYT450s) were responsible for the oxidation of the triterpenoid grams, suggesting consumption of Citrus fruit in daily diet might have precursor, which eventually lead to an array of limonoids. certain effect on human health. To date, about 55 limonoid aglycones (1–55, Fig. 3) and 18 limo- noid glycosides (56–73, Fig. 4) were identified from Citrus; the name, molecule formula, and source of those limonoids were summarized in 4. The biological activities of Citrus limonoids Table 1. For most Citrus species, limonin (26) is the most abundant aglycone, followed by nomilin (4), and limonin glucoside (72) is the Limonoids from Citrus were shown to possess an array of biological most representative limonoid glucoside (Tundis et al., 2014). Limonin activities, including antitumor, antioxidative, anti-inflammatory, neu- type is characterized by a tetrahydrofurano A-ring and a 3(4)-lactone roprotective, immunomodulatory, insecticidal, anti-bacteria, antiviral, with an epoxy-lactone D-ring. A six-membered lactone functional group anti-obesity, and anti-hyperglycemic activities. The pharmacological is connected with ring A through C-1 and C-10. Limonin (26)(Dreyer, activities that the representative limonoids demonstrate and biological 1965), limonexic acid (38)(Makita, Ohta, & Nakabayashi, 1980), iso- pathways that limonoids are involved in were illustrated in Fig. 5, and limonexic acid (40)(Lee, Morita, Takeya, Itokawa, & Fukaya, 1999), the detailed biological activities of these compounds were summarized limonol (24)(Bennett & Hasegawa, 1982), epilimonin (25)(Breksa, in Table 3.

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Fig. 2. Biosynthesis pathway of limonoids (Modified from the scheme by Nakatani, 2001).

4.1. Anti-tumor activity Sprague–Dawley rat model, limonin was shown to have protective ef- fect against azoxymethane-induced colon carcinogenesis measured at Limonoids obtained from Citrus plants were shown to exert in- the promotion stage (Fan et al., 2019). hibitory effects on the proliferation of colorectal, hepatocellular, gas- Likewise, obacunone and its glycosides were found to inhibit the tric, pancreatic, breast, and prostatic carcinoma, oophoroma, mela- proliferation of SW480 cells, with IC50 values of 97 µM and 109.7 µM, noma, neuroblastoma, and cheek pouch epidermoid carcinoma respectively at 24 h (Murthy, Jayaprakasha, & Patil, 2011; Murthy, (Gualdani et al., 2016; Kim, Jayaprakasha, Vikram, & Patil, 2012a, Jayaprakasha, Kumar, et al., 2011). 2012b). Isolimonic acid and ichanexic acid from Citrus aurantium L. were shown to have inhibitory activity on human colon cancer cells (HT-29): they can result in the significant arrest of cell growth within 24hat 4.1.1. Anti-colorectal carcinoma activity 5 µM and 10 µM, respectively; the treatment of both the compounds at Among limonoids, limonin was the most studied compound re- 5 µM caused nearly 4- to 5-fold increase in the number of G2/M stage garding anti-colorectal carcinoma activity. Limonin showed inhibitory cells, suggesting a potential role in the cell cycle arrest (Jayaprakasha activity on intestinal polyp development in Apc-mutant Min mice; li- et al., 2008). monin treatment reduced the expression levels of c-Myc and MCP-1 Limonexic acid was found to inhibit human colon cancer cell (HT- mRNA in the polyp part. Limonin was further found to have a role in β- 29) proliferation (66.5% inhibition at 40 μM after 72 h treatment), catenin signalling and showed a dose-dependent inhibitory effect on the while it exhibited no apparent toxicity on noncancerous cells at the transcriptional activity of T-cytokine/lymphocyte-enhancing factor in same concentration; the compound was demonstrated to be engaged in the Caco-2 human colon cancer cell line (Shimizu et al., 2015). In an- a highly promising arrest of the G2/M phase of the cell cycle resulting other report, limonin and limonin glucoside were found to exhibit in- in apoptosis (Jayaprakasha et al., 2010). hibition on human colon adenocarcinoma cells (SW480) with their IC50 Methyl nomilinate, isoobacunoic acid, isolimonexic acid, and li- value at 72 h of 54.74 and 37.39 μM, respectively; further mechanism monexic acid were assayed for their inhibition on SW480 human colon study indicated that both compounds inhibited SW480 cells in the in- adenocarcinoma cells: methyl nomilinate presented the most potent trinsic apoptosis pathway mediated through Bcl2 family protein and inhibition on cell metabolic activity in MTT and EdU incorporation cytochrome; additionally, the above compounds were also demon- assays, and methyl nomilinate exposure led to significant induction of strated to facilitate the apoptosis process by increasing intracellular 2+ G0/G1 cell cycle arrest. Moreover, methyl nomilinate repressed CDK4/ Ca concentration (Murthy, Jayaprakasha, & Patil, 2011; Murthy, 6 and cyclin D3 and the expression of CDK inhibitors, indicating that Jayaprakasha, Kumar, Rathore, & Patil, 2011). In a male

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Fig. 3. Chemical structures of limonoid aglycones from Citrus (1–55). the inhibition was mediated by G1 cell cycle arrest (Kim et al., 2012a, inhibition (up to 96%) of cell proliferation by apoptosis pathway 2012b). compared with the treatment of every single tested compound; the Intriguingly, treatment of combinations of limonoids and curcumin synergistic inhibition mode of limonoids and curcumin was further (final concentration = 50 ppm) on SW480 cells resulted in stronger supported by total caspase-3 activity and fluorescence microscopy of

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Fig. 3. (continued)

SW480 cells treated with limonoids, curcumin and combination other limonoid glucosides within the juice were considered to con- (Murthy, Jayaprakasha, & Patil, 2013). tribute to the decrease in colon carcinogenesis (Miyagi, Om, Chee, & Feeding single-concentration and pasteurized orange juice was Bennink, 2000). The subsequently in-depth study found that the dietary shown to inhibit the colon cancer induced by oxidized azomethane exposure to either obacunone or limonin in F344 rats model could re- (AOM) in male Fischer 344 rats; limonin 17-β-D-glucopyranoside and duce risk in the development of AOM-induced colonic adenocarcinoma:

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Fig. 3. (continued) the consumption of obacunone or limonin was shown to reduce the agents against colorectal carcinoma. incidence of colonic adenocarcinoma (72% versus 25% and 72% versus 6%, respectively during the initiation phase; 72% versus 13% for both 4.1.2. Anti-hepatocellular carcinoma activity compounds during the post initiation phase). Limonin was shown to possess weak inhibition activity on the The above investigations indicated that various limonoids such as proliferation of hepatocellular carcinoma cells (SNU-449) by the limonin, and obacunone possessed chemoprotective effect against colon apoptosis pathway (Rahman, Siddiqui, Jakhar, & Kang, 2015). Limonin cancer in both cell experiments and animal model, and the effect was was also found to inhibit the proliferation of Hep3B and HepG2 cells mainly mediated by the apoptosis pathway (Kaur & Kaur, 2015). (47.8% and 56.3% inhibition rate at 100 μM, respectively), and clone Therefore, Citrus limonoids could potentially be chemopreventive formation was also suppressed at the same concentration, being 86.7%

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Fig. 4. Chemical structures of limonoid glucosides from Citrus (56–73). and 83.3% for Hep3B and HepG2 cells, respectively. Further, limonin nitrosodiethylamine (DEN) induced phenobarbital promoted experi- was found to inhibit the tumor glycolysis in hepatocellular carcinoma mental hepatocellular carcinoma in male Wistar albino rats. By in- by suppressing HK-2 activity (Yao, Liu, & Zhao, 2018). Limonin was hibiting LPO and oxidative stress mediated free radicals generation and also demonstrated to possess protective effect against N- regulating antioxidants defense, limonin was shown to have good

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Table 1 Limonoids from Citrus (1–73), their molecule formula, and sources.

No. Compound Name Formula Source Reference

1 Obacunone C26H30O7 Citrus lemon (Kim et al., 2014)

2 7α-obacunol C26H32O7 Citrus paradisi (Bennett & Hasegawa, 1982)

3 7α-obacunyl acetate C28H34O8 Citrus-Poncirus hybrid (Bennett & Hasegawa, 1982)

4 Nomilin C28H34O9 Citrus iyo hort. ex Tanaka (Ishii et al., 2003)

5 Deacetylnomilin C26H32O8 Citrus pyriformis Hassk. (Hamdan et al., 2011)

6 6-keto-7β-deacetylnomilol C26H32O9 Citrus reticulata var. Austera × Fortunella sp. (Bennett & Hasegawa, 1981)

7 6-keto-7β-nomilol C28H34O10 Citrus reticulata var. Austera × Fortunella sp. (Herman et al., 1987)

8 nomilinic acid C28H36O10 Citrus sudachi (Hashinaga et al., 1990)

9 methyl nomilinate C29H38O10 Citrus aurantium (Bennett & Hasegawa, 1980)

10 deacetylnomilinic acid C26H34O9 Citrus aurantium (Bennett & Hasegawa, 1980)

11 methyl deacetylnomilinate C27H36O9 Citrus reticulata var. Austera × Fortunella sp. (Bennett & Hasegawa, 1981)

12 Calamin C27H36O10 Citrus reticulata var. Austera × Fortunella sp. (Bennett & Hasegawa, 1981)

13 Retrocalamin C24H30O9 Citrus reticulata var. Austera × Fortunella sp. (Bennett & Hasegawa, 1981)

14 obacunoic acid C26H32O8 Citrus paradise (Bennett et al., 1989)

15 isoobacunoic acid C26H32O8 Citrus paradise (Bennett et al., 1989)

16 methyl isoobacunoate C27H34O8 Citrus paradise (Bennett et al., 1989)

17 epiisoobacunoic acid C26H32O8 Citrus paradisi (Bennett et al., 1989)

18 methyl isoobacunoate diosphenol C27H32O9 Citrus reticulata var. Austera × Fortunella sp. (Bennett & Hasegawa, 1981)

19 Cyclocalamin C27H34O9 Citrus reticulata var. Austera × Fortunella sp. (Bennett & Hasegawa, 1981)

20 isocyclocalamin C27H34O9 Citrus reticulata var. Austera × Fortunella sp. (Herman et al., 1987)

21 limonoic acid C26H34O10 Citrus limon Burm. f. (Baldi, Rosen, Fukuda, & Ho, 1995)

22 limonoic acid D-ring lactone C26H32O9 Citrus paradisi (L.) Osbeck (Maier & Margileth, 1969)

23 limonilic acid C26H30O9 Citrus sp. (Pettit et al., 1983)

24 Limonol C26H32O8 Citrus paradisi (Bennett & Hasegawa, 1982)

25 Epilimonin C26H30O8 Citrus sp. (Citrus molasses) (Breksa et al., 2008)

26 Limonin C26H30O8 Citrus Evodia, Dictamnus and Luvunga sp. (Dreyer, 1965)

27 Deoxylimonol C26H32O7 Citrus paradisi (Bennett & Hasegawa, 1982)

28 Deoxylimonin C26H30O7 Citrus paradisi (Bennett & Hasegawa, 1982)

29 limonoic acid A-ring lactone C26H32O9 Citrus paradisi (Maier & Margileth, 1969)

30 17-dehydrolimonoate A-ring lactone C26H30O9 Citrus paradisi (Hsu, Hasegawa, Maier, & Bennett, 1973)

31 isolimonic acid C26H32O9 Citrus aurantium L. (Jayaprakasha et al., 2008)

32 Ichangin C26H32O9 Citrus limon, C. paradisi, and C. reticulata (Ozaki, Fong, et al., 1991)

33 Ichangensin C25H32O7 Citrus ichangensis (Bennett, Herman, & Hasegawa, 1988)

34 1-O-methylichangensin C26H34O7 Citrus sudachi (Nakagawa, Duan, & Takaishi, 2001)

35 1(10 → 19) abeo-obacun-9(11)-en-7α-yl acetate C28H32O8 Citrus-Poncirus hybrid (Bennett & Hasegawa, 1982)

36 1(10 → 19) abeo-7α-acetoxy-10β-hydroxyisoobacunoic acid C28H34O9 Citrus-Poncirus hybrid (Bennett & Hasegawa, 1982) 3,10-lactone

37 deoxylimonic acid C26H32O8 Citrus microcarpa (Hasegawa, Bennett, & Verdon, 1980)

38 limonexic acid C26H30O10 Citrus natsudaidai (Makita et al., 1980)

39 (-)-21-O-Methyllimonexic acid C27H32O10 Citrus reticulata (Khalil, Maatooq, & El Sayed, 2003)

40 isolimonexic acid C26H30O10 Citrus nippokoreana (Lee et al., 1999)

41 21,23-dihydro-23-methoxy-21-oxolimonin C27H32O10 Citrus reticulata Blanco (Kikuchi et al., 2017)

42 21,23-Dihydro-21-oxolimonin C26H30O9 Citrus reticulata Blanco (Kikuchi et al., 2017)

43 Citrobilin C28H34O11 Citrus nobilis (Bui, Duong, Tran, & Seip, 2004)

44 sudachinoid A C26H34O9 Citrus sudachi (Nakagawa et al., 2001)

45 sudachinoid B C25H32O9 Citrus sudachi (Nakagawa et al., 2001)

46 Citrusin C28H34O11 Citrus medica L. var. sarcodactylis swingle (Chan et al., 2010)

47 21,23-dihydro-23-hydroxy-21-oxodeacetylnomilin C26H32O10 Citrus sudachi (Nakagawa et al., 2006)

48 kihadanin B C26H30O9 Citrus natsudaidai (Makita et al., 1980)

49 21,23-dihydro-23-methoxy-21-oxonomilin C29H36O11 Citrus reticulata Blanco (Kikuchi et al., 2017)

50 21,23-Dihydro-21-hydroxy-23-oxonomilin C28H34O11 Citrus reticulata Blanco (Kikuchi et al., 2017)

51 sudachinoid C C26H30O9 Citrus natsudaidai (Makita et al., 1980)

52 21,23-dihydro-21-hydroxy-23-oxonomilinic acid methyl ester C29H38O12 Citrus reticulata Blanco (Kikuchi et al., 2017)

53 3-O-methyl-21,23-dihydro-23-hydroxy-21-oxonomilinic acid C29H38O12 Citrus sudachi (Nakagawa et al., 2006)

54 ichanexic acid C26H32O11 Citrus aurantium L. (Jayaprakasha et al., 2008)

55 citriolide A C25H28O6 Citrus reticulata Blanco (Liao, Xu, Liu, & Wang, 2012)

56 methyl deacetylnomilinate 17-β-D-glucopyranoside C33H48O15 Citrus reticulata var. austera × Fortunella sp. (Miyake, Ozaki, et al., 1992)

57 nomilinic acid glucoside C34H48O16 Citrus unshiu (Sawabe et al., 1999)

58 methyl nomilinate 17-β-D-glucopyranoside C35H50O16 Citrus unshiu (Sawabe et al., 1999)

59 calamin 17-O-β-D-glucopyranoside C33H48O16 Citrus reticulata var. austera × Fortunella sp. (Miyake, Ozaki, et al., 1992)

60 19-hydroxydeacetylnomilinic acid glucoside C32H46O16 Citrus aurantium (Miyake, Ayano, Ozaki, Herman, & Hasegawa, 1992)

61 obacunoic acid 17-O-β-D-glucopyranoside C32H44O14 Citrus paradisii (Bennett et al., 1989)

62 trans-obacunoic acid 17-O-β-D-glucopyranoside C32H44O14 Citrus paradisii (Bennett et al., 1989)

63 6-keto-7β-deacetylnomilol 17-O-β-D-glucopyranoside C32H44O15 Citrus reticulata var. austera × Fortunella sp. (Miyake, Ozaki, et al., 1992)

64 deacetylnomilin 17-β-D-glucoside C32H44O14 Citrus limon (Hasegawa et al., 1989)

65 obacunone 17-O-β-D-glucoside C32H42O13 Citrus limon (Hasegawa et al., 1989)

66 nomilin 17-O-β-D-glucopyranoside C34H46O15 Citrus limon (Hasegawa et al., 1989)

67 deacetylnomilinic acid 17-O-β-D-glucoside C32H46O15 Citrus paradisii (Bennett et al., 1989)

68 isoobacunoic acid 17-O-β-glucoside C32H44O14 Citrus paradisii (Bennett et al., 1989)

69 epiisoobacunoic acid 17-O-β-glucoside C32H44O14 Citrus paradisii (Bennett et al., 1989)

70 ichangin 17-β-D-glucopyranoside C32H44O15 Citrus hanaju (Ohta, Berhow, Bennett, & Hasegawa, 1992)

71 isolimonic acid 17-β-D-glucopyranoside C32H44O15 Citrus hanaju (Ohta et al., 1992)

72 limonin 17-β-D-glucopyranoside C32H42O14 Citrus limon (Hasegawa et al., 1989) (continued on next page)

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Table 1 (continued)

No. Compound Name Formula Source Reference

73 ichangensin 17-β-D-glucopyranoside C31H44O13 Citrus junos, Citrus sudachi and Citrus (Ozaki, Miyake, et al., 1991) sphaerocarpa

Table 2 4.1.3. Anti-gastric carcinoma activity Concentration values (mg/kg) of limonoid content in Citrus bergamia fruits. Up-regulation of detoxification pathway by the phase-Ⅱ enzymes

Compounds Juice Pulp Peel Seed such as Glutathione S-transferase (GST) and NAD(P)H: quinine re- ductase (QR) for the removal of various toxic compounds, such as Deacetyl nomilinic acid glucoside 8.3 229.1 – – carcinogens, chemotherapeutic drugs, environmental pollutants, and Limonin glucoside 7.1 6.2 12.3 16.5 oxidative stress products, is one of cellular protection mechanism that Deacetyl nomilin glucoside 11.3 – 44.5 114.9 may be anticarcinogenic (Dasari, Ganjayi, Yellanurkonda, Basha, & Nomilin glucoside 22.4 62.0 56.0 1651.9 Nomilinic acid glucoside 6.3 63.2 35.2 289.8 Meriga, 2018). Obacunone glucoside – 31.0 < LOQ < LOQ The effect of Citrus limonoids (nomilin, deacetyl nomilin, and iso- Ichangin – – 2.9 – obacunoic acid) and a mixture of them on phase II enzyme activity in Obacunoic acid – 24.2 29.3 83.2 excised stomach tissues of mice model was evaluated. The induction of Limonin 22.3 23.0 230.5 2098.1 Nomilin 13.1 75.5 394.1 2573.7 GST against 1-chloro-2,4-dinitrobenzene (CDNB) was detected in the All 90.8 514.2 804.8 6828.1 stomach (58% by nomilin, 25% by isoobacunoic acid, and 19% by deacetyl nomilin, respectively). Moreover, nomilin significantly in- duced GST potency against 4-nitroquinoline 1-oxide (4NQO) in the antioxidant and therapeutic property against DEN-induced hepato- stomach (75%). A significant induction of GST and NAD(P)H: QR ac- carcinogenesis (Langeswaran, Kumar, Perumal, Revathy, & tivities in the stomach was also observed (45% and 200%, respectively) Balasubramaniam, 2013). However, there have been no reports on the after the limonoid mixture treatment; these results suggest that Citrus anti-hepatocellular carcinoma activity of other Citrus limonoids, and limonoids may possess protective function against the onset of gastric more attention may be paid to those limonoids to probe their efficacies carcinoma by increasing the activity of some phase II detoxifying en- and structure and activity relationship. zymes in the stomach (Perez et al., 2010). The effectiveness of limonin and nomilin on Benzo(a)pyrene (BP)-

Fig. 5. The bioactivities (A) and the proposed mode of action (B) of Citrus limonoids.

9 Y.-S. Shi, et al. Journal of Functional Foods 75 (2020) 104213

Table 3 Biological activity of limonoids.

Compound Structure Reported biological activity Potency (in μM, unless otherwise stated)

Limonin Cytotoxicity in SW480 cells (Murthy, Jayaprakasha, & Patil, 54.74 at 72 h (IC50) 2011; Murthy, Jayaprakasha, Kumar, et al., 2011) Inhibitory activity on intestinal polyp development in Apc- 74% of the untreated control value (the mutant Min mice (Shimizu et al., 2015) total number of polyps in mice) Protective effect against azoxymethane-induced colon carcinogenesis (Fan et al., 2019) Inhibitory activity on proliferation of hepatocellular carcinoma cells (SNU-449) (Rahman et al., 2015) Cytotoxicity in Hep3B and HepG2 cells (Yao et al., 2018) 47.8% at 100 (Hep3B cells) and 56.3% at 100 (HepG2 cells) Protective effect against DEN-induced experimental hepatocellular carcinoma (Langeswaran et al., 2013) Cytotoxicity in MCF-7 cells (Somasundaram et al., 2012) GST inducing activity (Ahmad et al., 2006) 2.2 folds to control in the stomach Protective effect against DMBA-induced hamster buccal pouch 60% reduction in tumor burden epidermoid carcinomas (Miller et al., 1989) Inhibitory activities on melanogenesis in B16 cells (Akihisa 47% at 100 et al., 2017) Cytotoxicity in IOMM-Lee, CH157MN cells (Das et al., 2015) 40% at 25 (IOMM-Lee cells) and 41% at 25 (CH157MN cells)

Cytotoxicity in Panc-28 cells (Patil et al., 2010) 42.4 after 72 h (IC50) Inhibitory effect on P-glycoprotein in CEM/ADR5000 cells (El- Readi et al., 2010)

Inhibitory activity on CYP3A4 isoenzymes (Han et al., 2011)) 6.20 (IC50, testosterone as substrate), 19.1

(IC50, midazolam as substrate) Protective effect against experimentally-induced hepatic ischemia reperfusion (I/R) injury in rats (Mahmoud, Gamal, et al., 2014) Protective effect against D-galactosamine (D-GalN)-induced liver toxicity (Mahmoud, Hamdan, et al., 2014) Inhibitory effect on p38 MAP kinase in human aortic smooth 19% at 50 muscle cells (Kim et al., 2011) Antinociceptive activity (Matsuda et al., 1998))

Neuroprotective activity against glutamate-induced cytotoxicity 0.018 (EC50) in primary cultures of rat cortical cells (Yoon et al., 2008) inhibitory activity on NF-κB p65 nuclear translocation in activated CD4(+) T-cells in DO11.10 transgenic mouse model Antifeedant activity against Spodoptera frugiperda, Eldana saccharina, Maruca testulalis, and Leptinotarsa decemlineata (Bentley et al., 1988)

Moult inhibiting activity against Culex quinquefasciatus 59.57 ppm (EC50) (Jayaprakasha et al., 1997) Antifeedant activity against Spodoptera frugiperda (Ruberto 87 (feeding index) et al., 2002) Larvicidal activity against A. albopictus (Hafeez et al., 2011) 850.09, 600.72 and 407.09 at 24, 48 and

72 h (LC50)

Inhibitory activity HTLV-1 and HIV-1 expression (Balestrieri 1.07 μg/ml (IC50, HTLV-1) and 0.92 μg/ml

et al., 2011) (IC50, HIV-1)

Inhibitory activity on HIV-1 replication in PBMC system 60.0 (EC50) (Battinelli et al., 2003) Shorten the sleeping time of mice induced by α-chloralose and urethane (Wada et al., 1992)

Limonin 17-β-D- Cytotoxicity in SW480 cells (Murthy, Jayaprakasha, & Patil, 37.39 at 72 h (IC50) glucopyranoside 2011; Murthy, Jayaprakasha, Kumar, et al., 2011)

Cytotoxicity in Panc-28 cells (Patil et al., 2010) 20.49 after 72 h (IC50) Protective effect against DMBA-induced hamster buccal pouch 55% decrease in average tumor burden epidermoid carcinomas (Miller et al., 1992) compared to control

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10 Y.-S. Shi, et al. Journal of Functional Foods 75 (2020) 104213

Table 3 (continued)

Compound Structure Reported biological activity Potency (in μM, unless otherwise stated)

Obacunone Cytotoxicity in SW480 cells (Murthy, Jayaprakasha, & Patil, 97 at 24 h (IC50) 2011; Murthy, Jayaprakasha, Kumar, et al., 2011) Protective effect against AOM-induced colonic adenocarcinoma in F344 rats (Miyagi et al., 2000)

Cytotoxicity in ER+ human breast cancer cells (Guthrie et al., 0.009 μg/mL (IC50) 2000) Cytotoxicity in MCF-7, MDA-MB-231 cells (Kim et al., 2013) 44% at 200 (MCF-7 cells) and 18% at 200 μM (MDA-MB-231 cells)

Inhibitory activity on aromatase in MCF-7 cells (Kim et al., 28.04 (IC50) 2014) Protective effect against DMBA-induced hamster buccal pouch 25% reduction in tumor number and 40% epidermoid carcinomas (Miller et al., 2004) reduction in tumor burden Cytotoxicity in LNCaP cells (Murthy et al., 2015) about 80% at 100 mM at 48 h

Neuroprotective activity against glutamate-induced cytotoxicity 0.039 (EC50) in primary cultures of rat cortical cells (Yoon et al., 2008)

Insecticidal activity on Culex quinquefasciatus (Jayaprakasha 6.31 ppm (EC50) et al., 1997) Antifeedant activity against Spodoptera frugiperda (Ruberto 68 feeding index (Spodoptera frugiperda) et al., 2002) 1 mg/mL after 20 days (Mythimna separata) Insecticidal activity against Mythimna separata (Yu et al., 2015) 23.3% mortality at 1 mg/mL after 20 days Inhibitory activity on the biofilm formation and TTSS (Vikram et al., 2010), Antivirulence effect against S. Typhimurium (Vikram, Jayaprakasha, et al., 2012)

Obacunone 17-β-D- Cytotoxicity in SW480 cells (Murthy, Jayaprakasha, & Patil, 109.7 at 24 h (IC50) glucopyranoside 2011; Murthy, Jayaprakasha, Kumar, et al., 2011) Cytotoxicity in LNCaP cells (Murthy et al., 2015) 82% at 100 mM after 48 h

Cytotoxicity in SH-SY5Y cells (Poulose et al., 2006)) 5.54 (IC50)

Isolimonic acid Cytotoxicity in HT-29 cells (Jayaprakasha et al., 2008) about 50% at 5 Inhibitory effect on cell–cell signalling and biofilm formation in Vibrio harveyi (Vikram et al., 2011) Inhibitory effect on EHEC biofilm formation and attachment of EHEC to Caco-2 cells (Vikram, Jesudhasan et al., 2012)

Ichanexic acid Cytotoxicity in HT-29 cells (Jayaprakasha et al., 2008) about 65% at 5

Limonexic acid Cytotoxicity in HT-29 cells (Jayaprakasha et al., 2010) 66% at 40 after 72 h Cytotoxicity in SW480 cells (Kim et al., 2012a, 2012b)

Anti-pancreatic cancer (Panc-28 cells (Patil et al., 2010) 21.91 after 72 h (IC50, Panc-28 cells)

Antiviral (2.2.15 cells (Zhao et al., 2012) 39.22 µg/mL (IC50, HBsAg in 2.2.15 cells)

68.37 µg/mL (IC50, HBeAg in 2.2.15 cells)

Methyl nomilinate Cytotoxicity in SW480 cells (Kim et al., 2012a, 2012b) less than20% at 50 µM after 48 h

Cytotoxicity in ER+ human breast cancer cells (Guthrie et al., 0.01 μg/mL (IC50) 2000)

Isoobacunoic acid Cytotoxicity in SW480 cells (Kim et al., 2012a, 2012b) less than20% at 50 µM after 48 h

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11 Y.-S. Shi, et al. Journal of Functional Foods 75 (2020) 104213

Table 3 (continued)

Compound Structure Reported biological activity Potency (in μM, unless otherwise stated)

Isolimonexic acid Cytotoxicity in SW480 cells (Kim et al., 2012a, 2012b) about 20% at 50 µM after 48 h

Cytotoxicity in Panc-28 cells (Patil et al., 2010) 18.01 after 72 h (IC50)

Nomilin GST inducing activity in excised stomach tissues of mice model 58% GST induction compared to control (Perez et al., 2010) Protective effect against Benzo(a)pyrene (BP)-induced gastric 28% reduction in the number of mice tumors in female ICR/Ha mice (Lam & Hasegawa, 1989) identified with gastric tumors GST inducing activity (Ahmad et al., 2006) 2.96 folds to control in the stomach Inhibitory effect on p38 MAP kinase in human aortic smooth 38% at 50 muscle cells (Kim et al., 2011) Inhibitory activity on TNF-α-induced proliferation of HASMCs (Kim et al., 2017) Immunomodulatory activity on Balb/c mice (Raphael & Kuttan, 2003)

Moult inhibiting activity against Culex quinquefasciatus 26.61 ppm (EC50, Culex quinquefasciatus) (Jayaprakasha et al., 1997) Antifeedant activity against Spodoptera frugiperda (Ruberto 56 (feeding index) et al., 2002) Larvicidal activity against A. albopictus (Hafeez et al., 2011) 305.83, 176.08 and 136.07 at 24, 48 and

72 h (LC50, A. albopictus)

Inhibitory activity on HIV-1 replication in PBMC system 52.2 (EC50) (Battinelli et al., 2003) Potential in prevention for bone metabolic diseases (Kimira et al., 2015) Anti-obesity and anti-hyperglycemia activity (Ono et al., 2011) Shorten the sleeping time of mice induced by α-chloralose and urethane (Wada et al., 1992) Deacetyl nomilin GST inducing activity in excised stomach tissues of mice model 19% GST induction compared to control (Perez et al., 2010))

Anti-breast carcinoma (ER− and ER+ human breast cancer 0.07 μg/mL (IC50, ER− cells) 0.005 μg/mL

cells (Guthrie et al., 2000)) (IC50, ER+ cells) Inhibitory effect on p38 MAP kinase in human aortic smooth 19% at 50 muscle cells (Kim et al., 2011)

Isoobacunoic acid GST inducing activity in excised stomach tissues of mice model 25% GST induction compared to control (Perez et al., 2010)

Deacetylnomilinic acid GST inducing activity in the stomach of female A/J mice (Perez 55% GST induction compared to control glucoside et al., 2010) Inhibitory effect on cell–cell signalling and biofilm formation in Vibrio harveyi (Vikram et al., 2011)

Deoxylimonin Protective effect against DMBA-induced hamster buccal pouch 30% reduction in tumor number and 50% epidermoid carcinomas (Miller et al., 2004) reduction in tumor burden

Nomilinic acid glucoside Inhibitory activity on CYP isoenzymes (CYP1B1, CYP1B1, less than10 for each isoenzymes (IC50) CYP1A2, CYP3A4, and CYP19) (Poulose et al., 2007)

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12 Y.-S. Shi, et al. Journal of Functional Foods 75 (2020) 104213

Table 3 (continued)

Compound Structure Reported biological activity Potency (in μM, unless otherwise stated)

Ichangin Inhibitory effect on cell–cell signalling and biofilm formation in Vibrio harveyi (Vikram et al., 2011) Inhibitory effect on EHEC biofilm formation and attachment of EHEC to Caco-2 cells (Vikram, Jesudhasan et al., 2012)

Kihadanin B Inhibitory activity on adipogenesis in mouse 3 T3-L1 adipocytes (Baba et al., 2016)

7β-obacunol Shorten the sleeping time of mice induced by α-chloralose and urethane (Wada et al., 1992)

7α-limonol Shorten the sleeping time of mice induced by α-chloralose and urethane (Wada et al., 1992)

7β-limonol Shorten the sleeping time of mice induced by α-chloralose and urethane (Wada et al., 1992)

induced gastric tumors were also studied: the activity of GST in the liver 4.1.4. Anti-breast carcinoma activity of female ICR/Ha mice increased after treatment with nomilin in a limonin was shown to alone display inhibition on MCF-7 cell growth dose-dependent manner (2.48 and 3.44 times for 5 and 10 mg per an- and did not inhibit camptothecin-induced apoptosis; limonin demon- imal, respectively), while limonin did not show apparent activity. strated antitumor effect in a p53-dependent signalling network invol- Consequently, nomilin treatment reduced the number of mice identified ving the phosphorylations of ERK, p38 and both serine residues (Ser with gastric tumors from 100% to 72%, whereas limonin demonstrated 468 and Ser 536) of NFκB pathways as found in MCF-7 breast cancer less protective effect in the same case. Moreover, nomilin treatment cells but not in p53 mutant MDA-MB-231 cells. In vivo studies, limonin substantially reduced the number of tumors per mouse (Lam & addition through intraperitoneal injection substantially increased cy- Hasegawa, 1989). clophosphamide-induced tumor growth delay and decreased tumor Likewise, limonin and nomilin were shown to possess GST inducing weights according to the mitotic index (Somasundaram et al., 2012). capabilities (2.2 fold and 2.96 fold to control in the stomach, respec- 14 limonoids were tested for their inhibitory effect on the pro- tively) in another study (Ahmad et al., 2006). The stomach was the only liferation of estrogen receptor-negative (ER−) and -positive (ER+) organ among 4 organs tested in which the application of two limonoids human breast cancer cells. Among them, limonin methoxime and dea- led to a significant increase in GST activity. cetylnomilin exhibited the most potent inhibitory activity on ER− cells

Three Citrus limonoids (limonin, limonin glucoside, deacetylnomi- (IC50 = 0.02 and 0.07 μg/mL, respectively), while deacetylnomilin, linic acid glucoside, and two modified products of limonin, defuran obacunone and methyl nomilinate exerted the strongest inhibition on limonin and limonin 7-methoxime) were tested for their effect on phase ER+ cells ((IC50 = 0.005, 0.009, and 0.01 μg/mL, respectively) II enzymes in female A/J mice. Limonin-7-methoxime induced the GST (Guthrie et al., 2000). activity in the stomach by 51% compared to the control against 4-ni- 9 limonoids from seeds of Citrus lemon L. were tested for their in- troquinoline 1-oxide (4NQO), and the deacetylnomilinic acid glucoside hibitory effect on MCF-7 or MDA-MB-231 human breast cancer cells. treatment led to 55% GST induction in stomach homogenates (Perez Among them, obacunone possessed the most potent cytotoxicity at et al., 2010). 200 μM on MCF-7 cells (44% inhibition) and MDA-MB-231 cells (18% The above results in animal models all showed that the treatment of inhibition); further mechanism study indicated the antitumor effect on Citrus limonoids resulted in elevated GST activities, which was asso- MCF-7 cells was mediated by caspase-7 dependent pathways (Kim, ciated with the protective effect of these compounds against gastric Jayaprakasha, & Patil, 2013). The further in-depth study on the anti- carcinoma. Given strong GST induction activity of Citrus limonoids and proliferation effect of obacunone against MCF-7 cells revealed the their good selectivity towards the stomach, they may be good leads compound could up-regulate the expression of pro-apoptotic protein against gastric carcinoma. Bax, inhibit the expression of anti-apoptotic protein Bcl-2, and then

13 Y.-S. Shi, et al. Journal of Functional Foods 75 (2020) 104213 induce apoptosis. Simultaneously, obacunone induced cell cycle arrest melanogenesis-inhibitory activities (47% inhibition at 100 μM) without during the G1 phase of cell division. Additionally, obacunone showed apparent toxicity on B16 melanoma cells (79.6% cell viability) (Akihisa significant inhibition of aromatase activity, with50 anIC value of et al., 2017). 28.04 μM (Kim et al., 2014). Obacunone and obacunone glucoside were demonstrated to present The relationship between breast cancer resistance protein (BCRP/ time- and dose-dependent inhibitory activity against human androgen- ABCG2) and limonin was investigated using cell- and membrane-based dependent prostate cancer LNCaP cell proliferation (about 80% in- transport inhibition assays: limonin was shown to significantly inhibit hibition at 100 mM at 48 h); in addition to dose-dependent changes in methotrexate vesicle transport and ABCG2-mediated transport (Tan, Li, expression of proteins accountable for the induction of programmed Paxton, Birch, & Scheepens, 2013). cell death through the intrinsic pathway and down-regulation of Akt, The above studies showed that Citrus limonoids exerted inhibitory down-regulation of androgen receptor and prostate-specific antigen is effect on several breast cancer cell lines (MCF-7, ER−, ER+, MDA-MB- also involved in the cytotoxicity mechanism, which eventually resulted 231), with their potencies ranging from nanogram level to sub-milli- in activation of programmed cell death (Murthy, Jayaprakasha, & Patil, gram level, and the inhibition was shown to be mediated by many 2015). different pathways. Limonin was found to inhibit the growth of IOMM-Lee cells (about 40%) and CH157MN meningioma cells (about 41%) at 25 μM, while the 4.1.5. Anti-buccal pouch epidermoid carcinomas reference drug hydroxyurea showed less potency in the same case; it The effects of 12 limonoids on the development of 7,12- was further found that citrinin induced apoptosis with increased dimethyIbenz-[a]anthracene (DMBA)-induced buccal pouch epi- phosphorylation of glycogen synthase kinase 3β (GSK3β) by inhibition dermoid carcinomas using female Syrian hamsters were evaluated by of the Wnt5/β-catenin pathway. Moreover, no apoptosis was observed Hasegawa’s group (Miller et al., 1989). Among them, 4 Citrus limonoids in normal human neurons after limonin treatment (Das et al., 2015). were proved to exert protective effect against DMBA-induced buccal Human pancreatic cancer cells (Panc-28) were used to evaluate the pouch epidermoid carcinomas: limonin topical treatment on the animal anticancer potential of 5 limonoids: isolimonexic acid possessed the led to 60% reduction in tumor burden due to a 50% decrease in tumor most significant inhibitory effect on proliferation of Panc-28 cells mass and a 20% decrease in tumor number (Miller et al., 1989); limonin among these compounds (IC50 = 18.01 μM), followed by limonin 17-β-D-glucoside treatment resulted in a 55% decrease in average glucoside (IC50 = 20.49 μM), limonexic acid (IC50 = 21.91 μM), β- tumor burden mainly attributed to the reduction in tumor mass (Miller sitosterol glucoside (IC50 = 32.3 μM) and limonin (IC50 = 42.4 μM) et al., 1992); exposure to obacunone cause reduction in tumor number after 72 h of incubation. p21 dependent p53 and casapse-3 mediating and tumor burden by 25 and 40%, respectively; deoxylimonin treat- intrinsic apoptosis were shown to contribute to the observed cytotoxi- ment led to the decrease in tumor number and burden by 30 and 50%, city (Patil, Jayaprakasha, Murthy, Chetti, & Patil, 2010). respectively. However, nomilin and nomilin 17-β-D-glucopyranoside The anti-proliferative activity of limonoids (limonin, nomilin, no- showed weak activity, and limonol, nomilinic acid 17-β-D-glucopyr- milinic acid 17-O-β-D-glucopyranoside, obacunone 17-O-β-D-glucopyr- anoside, 17, 19-didehydrolimonoic acid, and deoxylimonic acid were anoside, and a limonoid glucoside mixture) against ovarian carcinoma inactive. Above result indicated that alteration in the A ring of the li- cells (SKOV-3) was tested. The limonoid glucoside mixture exhibited a monoid skeleton would result in a loss of anticancer activity, while partial inhibitory effect on SKOV-3 at 100 μg/ml. However, thefour changes in the D ring led to no apparent loss of biological activity individual limonoids had no significant impact on the growth of SKOV- (Miller et al., 2004). The above results suggested that Citrus limonoids 3 at the same concentration (Tian, Miller, Ahmad, Tang, & Patil, 2001). could be good candidates as chemopreventive agent against buccal The effects of limonin and deacetylnomilin on modulating theac- pouch epidermoid carcinoma. tivity of P-glycoprotein (P-gp) in the multidrug-resistant human leu- kaemia cell line (CEM/ADR5000) were investigated. Limonin and 4.1.6. Anti-neuroblastoma activity deacetylnomilin inhibited the efflux of the P-gp substrate rhodamine limonin, nomilin, deacetylnomilin, and obacunone were evaluated 123 in a dose-dependent manner. As a potent P-glycoprotein inhibitor, for their inhibitory activity on neuroblastoma cell line (SH-SY5Y). The limonin significantly enhanced doxorubicin cytotoxicity (2.98 fold and viability of SH-SY5Y cells was significantly reduced when exposed to 2.2 fold in Caco2 and CEM/ADR5000 cells, respectively) at a non-toxic the limonoids in a dose-dependent manner, while limonoid glucosides concentration of 20 μM. Limonin has been proved to be a good P-gp/ exerted more potent inhibition compared to aglycone. The inhibitory MDR1 reversal agent, which may improve the efficacy of chemotherapy effect of SH-SY5Y cells by treatment with glucosides had an increased agents (El-Readi, Hamdan, Farrag, El-Shazly, & Wink, 2010). ploidy compared with aglycones administration through flow cyto- Over-expression of CYP isoenzymes such as CYP1A2, CYP1B1, metric analysis, which was consistent with enhancing chromosomal CYP19 and CYP3A4 has been known to closely associated with the abnormalities (Poulose, Harris, & Patil, 2006). Further study on four onset of cancers of lung, breast, colon, and prostate. 8 limonoids were limonoid glucosides found that limoin 17β D-glucopyranoside and investigated for their effect on O-dealkylase and hydroxylase activities obacunone 17β D-glucopyranoside in micromolar range caused inhibi- of human cytochrome P450 (CYP) isoenzymes using ethoxyresorufin, tion of SH-SY5Y cell growth compared with untreated controls. In methoxyresorufin and dibenzylfluorescein as substrates: nomilinic acid contrast, nomilinic acid 17β D-glucopyranoside and deacetylnomilinic glycoside was the most potent inhibitor with an IC50 of less than 10 μM acid 17β D-glucopyranoside exerted weaker toxicity. Cytotoxicity was for different enzyme activities tested (CYP1B1, CYP1B1, CYP1A2, related to a concentration- and time-dependent increase in caspase 3/7 CYP3A4, and CYP19), indicating that nomilinic acid glycoside pos- activity, suggesting limonoid’s effect by apoptosis-inducing pathway, sessed a very substantial inhibitory effect on key enzymes that induce which was confirmed by flow cytometry and DNA fragmentation ana- carcinogenesis, and therefore are considered as potential anticancer lysis subsequently (Poulose, Harris, & Patil, 2005). candidates. (Poulose, Jayaprakasha, Mayer, Girennavar, & Patil, 2007). Likewise, limonin was shown to inhibit the activity of CYP3A4 iso-

4.1.7. Other antitumor activities enzymes (IC50 = 6.20 μM and 19.10 μM, with testosterone and mid- Limonoids from Citrus were shown to have inhibitory effects against azolam as substrates, respectively), whereas limonin exerted weak in- some other types of cancer, including melanoma, prostatic carcinoma, hibitory effects on human CYP1A2, CYP2C8, CYP2C9, CYP2C19, meningioma, pancreatic cancer, oophoroma, and leukaemia. CYP2D6, and P-gp (Han et al., 2011). 4 limonoids were tested for their melanogenesis inhibition effect in As shown above, limonoids presented inhibitory activity against the B16 melanoma cells induced by a-melanocyte-stimulating hormone various cell lines and demonstrated protective effect against several (a-MSH); among them, limonin exhibited the most potent cancers in animal models, while the anti-tumor effect was mediated by

14 Y.-S. Shi, et al. Journal of Functional Foods 75 (2020) 104213 multiple pathways. Compared with other food-derived compounds with inhibitor of p38 MAP kinase activity mediated by TNF-α in human reported anticancer activities such as curcumin (Moballegh Nasery aortic smooth muscle cells. The seven-membered A ring with the et al., 2020), zerumbone (Prasannan et al., 2012), escin (Tan et al., acetoxy group of nomilin was plausibly important in inhibiting the 2010), tocotreinols (Siveen et al., 2014) and thymoquinone (Siveen activity of p38 MAP kinase (Kim, Jayaprakasha, Muthuchamy, & Patil, et al., 2013), their overall moderate antitumor effect and good safety 2011). profile make Citrus limonoids have potential as chemopreventive agent The protective effectiveness of limonin on experimentally-induced against various cancers and merit more biological evaluation. hepatic ischemia reperfusion (I/R) injury in rats was investigated, which suggests that limonin possesses antioxidant and anti-in- 4.2. Antioxidative activity flammatory effects in ischemic liver and is consequently able to protect hepatocytes against I/R injury in rats. The observed antioxidant and Long-term overactivity of reactive oxygen species is harmful to the anti-inflammatory activity of limonin appeared to be mediated bythe function of cells and their crucial macromolecules, which includes the downregulation of the TLR-signalling pathway (Mahmoud, Gamal, & transformation of proteins into autoantigens and higher protein/DNA El-Fayoumi, 2014). Limonin was also shown to have a protective effect degradation. These issues ultimately lead to the occurrence and de- against D-galactosamine (D-GalN)-induced liver toxicity in a rat model velopment of many diseases, such as diabetes mellitus, cancers, and so of acute hepatic inflammation. Oral administration of limonin before D- on (Ighodaro, 2018; Samimi, Kalantari, Lorestani, Shirzad, & Saki, GalN injection, strongly attenuated markers of hepatic damage (ele- 2018; Sies, 2015). Phytochemicals and plant secondary metabolites vated liver enzyme activities and total bilirubin) and hepatic in- play a crucial role in the chemical prevention of diseases by inhibiting flammation (TNF-α, infiltration of neutrophils), oxidative stress and oxidative stress-induced DNA damage and regulate several oxidative expression of TLR-4 but not TLR-2 in D-GalN-treated rats (Mahmoud, stress-mediated signalling pathways through their antioxidant proper- Hamdan, Wink, & El-Shazly, 2014). ties (Andrisic et al., 2018; Chikara et al., 2018). Effect of limonin glucoside (LG) on circulating biomarkers of Citrus fruits were known for their antioxidant phytochemicals and chronic inflammatory diseases such as nonalcoholic fatty liver disease hence demonstrated potential in the prevention and treatment of var- (NAFLD), diabetes, CVD, and cancer were evaluated in a cross-over, ious chronic and degenerative diseases. While vitamin C is the most placebo-controlled, double-blind study in obese individuals. After the known antioxidant component in Citrus fruits, some limonoids within administration of LG, two markers of inflammation, MMP-9 and TNF-α, the fruits show comparable antioxidant potency to vitamin C: 4 limo- were reduced by 38.7% and 10.7%, respectively. Moreover, LG treat- noid glucosides were shown to possess O2− quenching activity, and ment substantially reduced concentrations of liver proteins: gamma- nomilinic acid 17β D-glucopyranoside demonstrated similar O2− glutamyl transferase (33.8%), alanine aminotransferase (13.1%), alka- quenching activity to vitamin C and was almost as potent as superoxide line phosphatase (10.1%), and complement C3 (6.4%), the elevation of dismutase in slowing the decomposition rate within the 5-min test time which were implicated with the onset of above diseases. Thus, LG may (Poulose et al., 2005). have potential application in the prevention of these diseases (Kelley In another study, vascular smooth muscle cells were pre-treated et al., 2015). with native low-density lipoprotein (nLDL), and incubated with limonin In the formalin test, limonin was shown to have antinociceptive subsequently. Mitochondrial reactive oxygen species (ROS) generation activity. Further, the increase of vascular permeability induced by was increased by nLDL stimulation, while limonin was shown to block acetic acid and the paw edema induced by carrageenin were suppressed reactive oxygen species generation (Koo et al., 2018). by limonin. Limonin also presented inhibition on bradykinin-induced The antioxidant activities of two Citrus limonoids, limonin and li- paw edema and arachidonic acid-induced ear swelling. These results monin 17-O-β-D-glucopyranoside, were evaluated using various in vitro provide evidence for the antinociceptive activity of limonin accom- models such as superoxide, 1,1-diphenyl-2-picryl hydrazyl (DPPH), β- panied with an anti-inflammatory effect (Matsuda, Yoshikawa, Iinuma, carotene-linoleic acid, and hamster low-density lipoprotein (LDL): & Kubo, 1998). compared with eight flavonoids tested, both limonoids showed weaker A series of water-soluble derivatives of limonin were synthesized antioxidant effect in all the models above (Yu et al., 2005). and tested in anti-inflammatory models: the most potent compound As above, Citrus limonoids were shown to have moderate/weak demonstrated a more substantial anti-inflammatory effect than the re- antioxidative activity in different models. However, more studies need ference drug naproxen. Therefore, limonin was demonstrated as a be to carried out to verify the antioxidative potential of other Citrus promising lead for anti-inflammatory drugs (Yang et al., 2014). limonoids since there were few researches concerning this. Nomilin was found to have an anti-proliferation effect on TNF-α- induced human aortic smooth muscle cells (HASMCs) mediated partly 4.3. Anti-inflammatory activity by suppressing inflammatory signalling. Nomilin substantially reduced the phosphorylation of IκBα, an inhibitor of NF-κB, and decreased the Limonoids demonstrated potential in the prevention of various downstream inflammatory signalling in TNF-α treated HASMCs suc- diseases through anti-inflammatory activities. Mitogen-activated pro- cessively (Kim, Chakraborty, Jayaprakasha, Muthuchamy, & Patil, tein (MAP) kinases are important players in cellular signalling path- 2017). ways and mediate a variety of cellular behaviours in response to ex- Overall, Citrus limonoids exerted their anti-inflammatory effect tracellular stimuli. The p38 MAP kinases, one of the sub-groups, serve through multiple pathways and had therapeutic potential in many as- as a signal transduction mediator and play a vital role in many biolo- sociated diseases such as hepatic ischemia reperfusion (I/R) injury, gical processes (Topolska-Wos, Rosinska, & Filipek, 2017). acute hepatic inflammation, and many other chronic inflammatory The mediatory roles on p38 MAP kinase activity by seven structu- diseases. rally different limonoids were investigated using vascular smooth muscle cells. Nomilin exhibited the highest inhibition (38%) of p38 4.4. Anti-neurological diseases MAP kinase activity, followed by limonin (19%), deacetyl nomilin (19%), and defuran nomilin (17%). However, methyl nomilinate and Obacunone and limonin were shown to have significant neuropro- defuran limonin treatment resulted in no significant decrease in p38 tective activity against glutamate-induced cytotoxicity in primary cul-

MAP kinase activity. Conversely, obacunone application increased the tures of rat cortical cells (EC50 = 0.039 and 0.018 μM, respectively). p38 MAP kinase activity significantly by 38%. Further, P38 MAP kinase Notably, both obacunone and limonin demonstrated better neuropro- activity induced by TNF-α in the smooth muscle cells was totally in- tective effect than the reference drug dizocipline maleate hibited by the nomilin treatment. Thus nomilin was an effective natural (EC50 = 0.48 μM), a noncompetitive antagonist of NMDA receptor, at a

15 Y.-S. Shi, et al. Journal of Functional Foods 75 (2020) 104213 concentration of 0.1 μM (Yoon, Sung, & Kim, 2008). insecticidal activity of nomilin and obacunone over limonin. Besides, The neuroprotective mechanism of limonin and obacunone against moult inhibition effect by three limonoids was also observed glutamate toxicity was investigated using primary cultured rat cortical (Jayaprakasha, Singh, Pereira, & Sakariah, 1997). In another model of cells. The pretreatment of both limonin and obacunone (working Spodoptera frugiperda (a commercially important pest) (Ruberto, Renda, concentration = 0.1 μM) effectively attenuated glutamate-induced Tringali, Napoli, & Simmonds, 2002), however, limonin demonstrated neurotoxicity with their cell viability of 65.8% and 68.3%, respectively, the most significant antifeedant potency (feeding index = 87), followed while the cell viability of glutamate-induced damaged group was 0%. by obacunone and nomilin (feeding index = 68 and 56, respectively), Both compounds were shown to significantly inhibit the increase of suggesting different behavioural responses of two insect species to Ca2+ in cortical cells damaged by glutamic acid and reduced the structural alteration. amount of glutamate-induced NO and reactive oxygen species produc- Obacunone was demonstrated to possess insecticidal activity against tion. Furthermore, limonin significantly up-regulated the mitochondrial Mythimna separata (Walker): it exhibited 23.3% mortality rate com- membrane potential and activities of antioxidative enzymes. Thus pared with 26.7% mortality of reference drug toosendanin at 1 mg/mL above compounds exerted their neuroprotective effect by protecting the after 20 days (Yu, Ding, Zhi, & Xu, 2015). antioxidant defense system and are demonstrated as good leads for Limonin and nomilin were tested for larvicidal activity against A. glutamate-related neurodegenerative diseases (Yoon, Yang, Kim, Sung, albopictus using a method recognized by the World Health & Kim, 2010). Organization: LC50 of compound limonin at 24, 48, and 72 h were 850.09, 600.72, and 407.09 µM, respectively, while nomilin presented

4.5. Immunomodulatory activity more substantial larvicidal effect with the corresponding LC50 of 305.83, 176.08, and 136.07 µM (Hafeez, Akram, & Shaalan, 2011). CD4(+) T cells play an important role in guiding the appropriate According to these studies, limonin, nomilin, and obacunone were immune response in host defense and the pathogenesis of inflammatory most investigated for their anti-insect activities, while their potency diseases. In addition to the classical two-phase model of T-helper 1 against different insects varied significantly. Considering their good (Th1) and Th2 cell differentiation, the number of CD4(+) T cell subsets activities against some of the representative insects (e.g. Mythimna se- increased unexpectedly (Hirahara & Nakayama, 2016). parata, Culex quinquefasciatus) and safety profile in human, Citrus li- The effects of limonin on NF-κB-dependent CD4(+) T-cell pro- monoids represent a class of anti-insect compounds worthy of further liferation was examined using DO11.10 transgenic mice. Limonin study. treatment was demonstrated to inhibit NF-κB p65 nuclear translocation in activated CD4(+) T-cells. However, limonin diets did not affect ac- 4.7. Antiviral activities tivator protein-1 or nuclear factor of activated T-cells c1. On the con- trary, interleukin-2 production was not directly related to NF-κB status. HIV infection can cause systemic T cell damage, which results in Additionally, dietary combination with 4% fish oil and 1% corn oil (FO) suppression in immunity. It directly damages many tissues and causes enhanced the inhibitory effects of limonin concerning CD4(+) T-cell systemic severe organ damage (Gao et al., 2019). Antiretroviral therapy proliferation in response to anti-CD3/28 mAb. These results indicated enables HIV-infected people to survive for a longer time, but there are that combination chemotherapy might be beneficial in regulating in- some side effects, including immune reconstitution inflammatory syn- flammation mediated by CD4(+) T-cell (Kim et al., 2009). drome induced response. Natural products may provide a good solution The effect of nomilin on the immune system was evaluated using for HIV treatment (Lucas & Nelson, 2015). Balb/c mice. Intraperitoneal injection of nomilin increased the total Studies on the effect of limonin and nomilin on the carcinogenic number of white blood cells (WBC). Compared with the control group, delta retrovirus human T-cell leukemia/lymphoma virus type 1 (HTLV- the total number of bone marrow cells and α-esterase positive cells 1) showed that the potency of limonin in inhibition of the expression of increased in nomilin treated groups. The specific immune response was HTLV-1 and HIV-1 in infected cells was close to that of the reference detected by spleen erythrocyte. The particular antibody titer and the drug nevirapine. Furthermore, limonin showed less cytotoxicity com- amount of plaque-forming cells in the spleen were increased by the pared to nevirapine. (Balestrieri et al., 2011). Limonexic acid was application of nomilin along with antigen. The lymphocyte count was shown to inhibit the expression of HBsAg (IC50 = 39.22 µg/mL) and decreased compared with healthy animals. Nomilin significantly in- HBeAg (IC50 = 68.37 µg/mL) in 2.2.15 cells, indicating limonexic hibited delayed-type hypersensitivity. The results showed that nomilin acid’s potential against hepatitis B virus (Zhao, Yang, Wei, Huang, & had remarkably immunomodulatory activity (Raphael & Kuttan, 2003). Jiang, 2012). The antiretroviral activities of limonin and nomilin against the 4.6. Anti-insect activity growth of HIV-1 were investigated using human peripheral blood mononuclear cells (PBMC) and monocytes/macrophages (M/M). The anti-insect potential of limonoids was recognized since azadir- Limonin and nomilin exhibited a dose-dependent inhibition of HIV-1 achtin, a limonoid possessing broad-spectrum antifeedant activity, was replication in PBMC system (EC50 = 60.0 μM and 52.2 μM, respec- isolated in 1968 (Fernandes et al., 2019). The anti-insect activity of tively). The two limonoids inhibited the production of HIV-p24 antigen limonoids of Citrus has been shown to be mainly mediated by their at the tested concentration (20–100 μM), even when the PBMC were insecticidal, insect antifeedant and growth-regulating effect. chronically infected. Furthermore, limonin and nomilin also sig- Early studies demonstrated that limonin displayed antifeedant ef- nificantly inhibited the replication of HIV-1 in infected M/M. The anti- fectiveness against several insect species such as Spodoptera frugiperda, HIV activity of limonin and nomilin was shown to be mediated by the Eldana saccharina, Maruca testulalis, and Leptinotarsa decemlineata inhibition of in vitro HIV-1 protease activity (Battinelli et al., 2003). (Bentley, Rajab, Alford, Mendel, & Hassanali, 1988). Further structure- As above, Citrus limonoids demonstrated antiviral activities against activity relationship study suggested the essentiality of the epoxide and several viruses including HIV-1, HTLV-1, and hepatitis B virus. furan groups for its antifeedant effect against L. decemlineata. However, their potencies were generally at the high micromolar level. Other limonoids from Citrus were later assayed for their anti-insect potential against several representative insect species. The inhibitory 4.8. Antibacterial activity effects on Culex quinquefasciatus instar larvae (vector of the pathogen transmission) by limonin, nomilin, and obacunone were studied, with Obacunone demonstrated potent inhibition of cell-to-cell signalling their EC50 determined to be 59.57, 26.61 and 6.31 ppm, respectively. of Escherichia coli O157:H7 (EHEC) mediated by AHL and AI-2, EHEC The 7-membered lactone was considered to be responsible for the better biofilm formation, and type three secretion system (TTSS).

16 Y.-S. Shi, et al. Journal of Functional Foods 75 (2020) 104213

Furthermore, obacunone and other limonoids seem to inhibit the bio- 5. Metabolic studies film formation and TTSS in quorum sensing dependent manner (Vikram, Jesudhasan, Jayaprakasha, Pillai, & Patil, 2010). Obacunone In addition to their various pharmacological properties, Citrus li- exerts an antivirulence effect on S. Typhimurium mediated by repres- monoids are commonly present in the daily diet, as limonoids exist in sing Salmonella pathogenic island 1 (a maltose transporter) and Sal- orange juice at the mean concentration of 320 mg/L (Fong, Hasegawa, monella pathogenic island 2 pathway (Vikram, Jayaprakasha, Herman, & Ou, 1989). Therefore, the investigation into the metabolic Jesudhasan, Pillai, & Patil, 2012). Ichangin, deacetylnomilinic acid fate of Citrus Limonoids by the human body is of great concern. Limonin glucoside, and isolimonic acid were demonstrated to possess a good glucoside makes up 56% of the average total limonoid glucoside mix- inhibitory effect on autoinducer-mediated cell–cell signalling and bio- ture in Citrus juice (Fong et al., 1989). In this light, limonin glucoside film formation in Vibrio harveyi, and ichangin and isolimonic acidwere was first evaluated for its absorption, metabolism, and bioavailability in further determined to exert the above effect by regulating the expres- the human body (Manners et al., 2003). Limonin glucoside was con- sion of the luxO gene (Vikram, Jesudhasan, Jayaprakasha, Pillai, & verted into limonin as the major product and another compound which Patil, 2011). Ichangin and isolimonic acid were also shown to inhibit was proposed to be epilimonin. When different amount of limonin EHEC biofilm formation and attachment of EHEC to Caco-2 cells, and (0.25–2 g) was applied, the mean concentration of limonin in human the further study suggested both compounds exhibited inhibition in plasma from all subject groups ranged from 1.74 to 5.27 nmol/L with biofilm formation and TTSS by suppressing LEE and flagellar operon the corresponding mean time being 6 h, indicating limonin glucoside is (Vikram, Jesudhasan, Pillai, & Patil, 2012). Those results demonstrated bioavailable. Furthermore, post-study in the health condition of the that limonoids exerted their antibacterial effect mainly mediated by subject showed that no adverse effect was observed after the treatment their antivirulence effect and the inhibition of cell-to-cell signalling and of limonin glucoside. In a 127-day cross-over, placebo-controlled, and biofilm formation instead of bacteriostasis or sterilization. double-blind study, limonin glucoside was further evaluated for its safety and biological activities on in 10 overweight/obese individuals 4.9. Other biological activities (Kelley et al., 2015): limonin glucoside was shown to remarkably lower the concentrations of liver proteins, while it exerted no specific side Animal experiment researches have revealed a positive association effect. Overall, the above studies emphasize the safe metabolic profile between Citrus fruits intake and bone health. The effects and me- and good bioavailability of limonin glucoside. chanism of nomilin on osteoclastic differentiation of mouse primary bone marrow-derived macrophages (BMMs) and the mouse RAW 264.7 6. Conclusion macrophage cell line into osteoclasts were investigated using in vitro cell cultures. It was found that nomilin significantly reduced the Limonoids, together with vitamin C, hesperidin, and other biologi- number of TRAP-positive multinucleated cells, bone resorption activity, cally active substances in Citrus fruits are of great significance in pro- the expression of some genes in osteoclasts, the levels of NFATc1 and moting health. Limonoids from Citrus have been shown to exhibit an TRAP mRNA, and inhibited the phosphorylation of ERK, p38, and JNK array of bioactivities including antitumor, antioxidative, anti-in- in the MAPK signalling pathways. These results suggested that nomilin flammatory, neuroprotective, immunomodulatory, insecticidal, anti- could inhibit the phosphorylation of MAPKs induced by RANKL and bacteria, antiviral, anti-obesity, and anti-hyperglycemic activities. significantly suppressed the differentiation of osteoclasts in vitro. Thus Moreover, their high abundance in dietary Citrus fruit and good toler- nomilin demonstrates potential application for prevention for bone ance by the human body provide even more promises for limonoids as metabolic diseases (Kimira et al., 2015). nutritional supplements, functional food, medications against various G protein-coupled receptors (GPCRs) are a prominent family of disease or chemopreventive agent. membrane receptors with essential physiological and pathological Despite attractive pharmacological properties of Citrus limonoids, functions (Hauser, Attwood, Rask-Andersen, Schioth, & Gloriam, 2017). there are several issues which hinder the pharmaceutical application of As a member of the GPCRs family, TGR5 is activated by bile acids. them. As limonoids shared with a complex triterpenoid scaffold with Because of the effects on the promotion of energy consumption and several chiral centers, the total synthesis of limonoids is quite chal- improvement of glucose metabolism, TGR5 showed anti-obesity and lenging so far (Fu & Liu, 2020). An alternative approach for limonoid anti-hyperglycemic activities in mice and was considered as a critical supply and diversification is semi-synthesis since significant quantities target for the treatment of metabolic diseases (Park, Lee, Kim, Seo, & of limonoids are available in Citrus peels and seeds used during the Chung, 2016). Nomilin was demonstrated to be an activator of TGR5 production of juices and jams and can also be obtained by the debit- (Sato, 2013). Mice treated with nomilin had lower body weight, serum tering process in Citrus juice industry. However, limonoids from these glucose, serum insulin, and increased glucose tolerance. The results sources are generally afforded through tedious procedures. For ex- suggested that nomilin was a potential agent having anti-obesity and ample, a recent study reported the isolation of limonin from Citrus anti-hyperglycemic activities, which was possible to be mediated by the grandis peel by water extraction (4.7 mg/g), ammonium sulfate pre- activation of TGR5 (Ono, Inoue, Hashidume, Shimizu, & Sato, 2011). cipitation (2.4 mg/g), resin adsorption, and crystallization, with an Researches indicated that the agonist effect of nomilin on mouse TGR5 overall low yield (Yang et al., 2017); other recently developed methods was much weaker than on human TGR5, which was demonstrated by also shared with the same issue (Xiang et al., 2014; Yu, Wang, Deng, & the use of the cAMP response element-luciferase reporting analysis Bi, 2017). Therefore, the development of convenient and efficient ways system in HEK293 cells. Additionally, three amino acid residues to obtain limonoids from Citrus could be an important scientific ques- (Q77ECL1, Y893.29, and R80ECL1) played an essential role in the in- tion for the latter research. Secondly, while Citrus limonoids possessed teraction of hTGR5 and nomilin (Sasaki et al., 2017). versatile pharmacological activities, especially their anti-tumor and Kihadanin B from the peels of immature Citrus unshiu was found to anti-inflammatory activities, their potencies are generally at ther- inhibit adipogenesis through suppression of the Akt-FOXO1-PPARγ axis apeutically unfavourable doses. Delightfully, Citrus limonoids have in mouse 3 T3-L1 adipocytes, indicating the anti-obesity potential of been proven to be well tolerated in vitro and in vivo, suggesting their this compound (Baba, Ueno, Kikuchi, Tanaka, & Fujimori, 2016). advantageous role as nutraceuticals. Thirdly, the detailed biological 6 limonoids (obacunone, 7β-obacunol, limonin, 7α-limonol, 7β-li- activities of Citrus limonoids in human body have been rarely reported monol and nomilin) were found to shorten the sleeping time of mice so far (Kelley et al., 2015; Manners et al., 2003) and are still required to induced by α-chloralose and urethane, and nomilin was shown to pre- validate their safety profile, bioavailability and pharmaceutical poten- sent the highest reduction rate of sleeping time (about 64%) (Wada, tial further. Yagi, Kurihara, & Haga, 1992). In this report, the chemical structure, the occurrence in different

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