A Review on Bioactive Compounds and Pharmacological Activities of Citrus unshiu

Abstract

Citrus constitutes a group of fruit crops that include lemons, limes, mandarins, oranges, and grapefruits. These citrus have a variety of essential nutrients and bioactive compounds that exert several pharmacological properties, including antioxidant, anti-inflammatory, anticancer, anti-diabetic, and anti-obesity. The Satsuma mandarin, also known as Citrus unshiu Marc. (C. unshiu), is one of the most popular citrus types. This is mostly due to its seedless nature, early maturity, and highly valued commercial trait in the citrus sector. The pharmacological properties and phytochemicals of the two main citrus fruits—sweet oranges (C. sinensis) and lemons (C. limon)—are given more attention. Satsuma mandarin has not, however, had its therapeutic properties thoroughly examined and explored. Therefore, the purpose of this review is to use multiple databases to compile the information currently available on the pharmacological properties and bioactive compounds of C. unshiu. The findings indicate that C. unshiu bioactives (hesperidin; hesperetin; nobiletin; narirutin; naringin; naringenin; tangeretin; 3,5,6,7,8,3′,4′-heptamethoxyflavone; neoponcirin; synephrine; quercetin; quercetagetin; rutin; β-cryptoxanthin; and pectin) exert in vitro/vivo anticancer, anti-obesity, anti-diabetic, cardioprotective, gastroprotective, neuroprotective, hepatoprotective, skin-protective, nasal airway-protective, lipid-lowering, antioxidant, anti-inflammatory, and anti-microbial activities. Future experimental investigations into the potential health benefits of C. unshiu could contribute to a better understanding of the mechanisms behind its therapeutic activities. Clinical studies are necessary to assess the therapeutic properties of C. unshiu bioactives. The therapeutic potential of C. unshiu bioactives should be determined through preclinical in vivo animal studies before they can be applied in clinical settings.

1. Introduction

Citrus fruits, including lemons (C. limon), limes (C. aurantifolia), sweet oranges (C. sinensis), sour oranges (C. aurantium), grapefruits (C. paradisi), and others, are flowering plants with different sizes and forms, which belong to the genus Citrus in the family Ructaceae [1]. These citrus fruits have been recognized as the most crucial for improving human health because they are a major source of vital nutrients and energy [1]. Citrus fruits are acknowledged for their anticancer, antioxidant, and anti-inflammatory activities due to the high levels of phytochemicals present in various parts of the plant, including the peels, seeds, and pulps [1,2,3,4,5,6]. The Satsuma mandarin, also called Citrus unshiu Marc. (C. unshiu), is unique among mandarins due to its high flavor, seedless nature, highest total phenolic content, antioxidant potential, and significant amounts of fragrance compounds [7].

About 50–60% of the entire fruit mass is made up of citrus peels (albedo and flavedo), seeds, and pulps, which are acquired as byproducts (citrus wastes) during the processing of citrus fruits [8]. The albedo is the internal white layer of the peel that contains pectin, vitamins, and flavanones (e.g., narirutin, hesperidin). The flavedo is the peel’s outer layer, which is rich in triterpenoids, pigments, steroids, and essential oils. Seeds are rich in fiber, crude protein, lipids, and nitrogen-free extract. Pulps mainly consist of organic acids, insoluble fiber, glucose, and fructose. The chemical constituents of citrus fruits and their byproducts are influenced by several factors, including extraction techniques/mode, storage period, cultivation method, stage of ripeness, and harvesting time [9,10,11,12,13].

Citrus flavonoids have low intestinal absorption, which has been linked to poor bioavailability in a number of experiments [14]. An in vivo experiment has shown a large amount of carotenoid β-cryptoxanthin (βCX) absorbed and stored in different rat tissues (liver, brain, heart, lung, spleen, kidney, testis) following administration of a standard diet containing C. unshiu [15].

A large body of evidence indicates that citrus essential oils used in cosmetic products are safe [16,17]. However, essential oils isolated from citrus fruits (e.g., grapefruit, bergamot) demonstrated cytotoxic effects against various cancers [16,18]. In vitro and in vivo experimental evidence indicates that C. unshiu peel exhibits no genotoxicity and minimal systemic toxicity [19]. A clinical investigation revealed that overweight or obese individuals can safely use C. unshiu peel pellets to improve their weight loss and lower their cholesterol and triglyceride levels [20]. According to a randomized placebo-controlled study, patients with periorbital wrinkles who received a safe dosage of green mandarin extract containing narirutin (300 mg/day) for 12 weeks had an improvement in skin roughness in comparison to those in the placebo group (no green mandarin extract) [21].

Experimental evidence shows that lemon (C. limon) and sweet orange (C. sinensis) bioactives (e.g., limonene, terpenes, hesperedin, naringin, daidzin, βCX) exerted various medicinal properties, including anti-diabetic, anti-obesity, and anticancer, through modulating signaling pathways as the mechanisms for these activities [22,23,24]. However, there has not been much focus on the pharmacological properties and mechanisms of action of C. unshiu bioactives. The main bioactive compounds found in C. unshiu, together with their therapeutic properties, have not, as far as we are aware, been reviewed. This paper thus provides a summary of studies currently available on the pharmacological properties, bioactive compounds, and therapeutic mechanisms of Satsuma mandarin (C. unshiu). This review made the assumption that C. unshiu is a significant source of many classes of bioactive compounds that, through various mechanisms of action, exhibit pharmacological activities in both in vitro and in vivo experiments.

2. Methods

Information on Satsuma mandarin or C. unshiu Marc. was retrieved from the PubMed/Medline and Scopus databases, using the following search terms: (C. unshiu AND botanical), (C. unshiu AND pharmacological), (C. unshiu AND phytochemistry), and (C. unshiu AND bioactivity). Studies were included if they focused on the extraction of bioactive compounds from C. unshiu. Experiments that investigated the therapeutic and/or medicinal activities of C. unshiu using in vitro and in vivo models were eligible. There were no restrictions on the publication date, and any papers published exclusively in English were eligible. A total of 85 studies published up until the end of January 2025 were obtained.

3. Botanical Description and Bioactive Compounds of C. unshiu Marc.

Satsuma mandarin (C. unshiu Marc.) is a widely cultivated citrus that originated in Southeast Asia (mainly in Korea and Japan) [25] and is considered the most adaptable and cold-resistant citrus with high commercial value [26,27]. Satsuma mandarin is often seedless [28], and has two tissue types: flesh (pulp or endocarp) and rind (peel or pericarp) [28,29]. The mandarin pulp consists of vascular bundles and segment membranes containing juice vesicles. The mandarin peel is divided into the external colored layer (flavedo or exocarp) and the internal white layer (albedo or mesocarp) (Figure 1).

Three different groups of flavonoids (flavanones, polymethoxylated flavones, and flavonols), carotenoid βCX, alkaloid synephrine, and dietary fiber pectin were isolated from Satsuma mandarin, as illustrated in

Table 1. Bioactive compounds isolated from Satsuma mandarin.

Bioactive Compounds	Part Used	Solvents	Extraction Methods	Bioactive Values	Ref.
Hesperidin	Peel	Methanol	Raman microscopy	Hesperidin: 64.3 mg/g	[30]
Hesperidin, narirutin, naringin, nobiletin, tangeretin	Peel, pulp	Methanol	High-performance liquid chromatography	Hesperidin: 51.9–73.8 mg/g; Narirutin: 8.0–19.8 mg/g; Naringin: 0.35–0.40 mg/g; Nobiletin: 0.2–0.6 mg/g; Tangeretin: 0.13–0.25 mg/g	[31]
Hesperidin, narirutin	Peel	Methanol, water	Subcritical water extraction	Hesperidin: 46.96 mg/g; Narirutin: 8.76 mg/g	[32]
Hesperidin, narirutin, rutin, heptamethoxyflavone	Peel	Water	High-performance liquid chromatography, supercritical CO2	Hesperidin: 0.16–15.07 mg/g; Narirutin: 0.11–4.87 mg/g; Rutin: 0.18–4.27 mg/g; HMF: 0.01–14.33 mg/g	[33]
Hesperidin, narirutin	Peel	Methanol	Microwave-assisted extraction	Hesperidin: 5860 mg/100 g; Narirutin: 1310 mg/100 g	[34]
Hesperidin, narirutin, naringin, nobiletin, rutin, tangeretin	Peel	Ethanol	High-performance liquid chromatography	Hesperidin: 4297 mg/100 g; Narirutin: 3469 mg/100 g; Naringin: 24.6 mg/100 g; Nobiletin: 107.5 mg/100 g; Rutin: 1098.6 mg/100 g; Tangeretin: 41.7 mg/100 g	[35]
Hesperidin, narirutin, nobiletin, rutin, tangeretin	Peel	Methanol	Ultra-high-performance liquid chromatography	Hesperidin: 16,522.2 mg × kg−1; Narirutin: 8686.6 mg × kg−1; Nobiletin: 759.9 mg × kg−1; Rutin: 3068.7 mg × kg−1; Tangeretin: 311.4 mg × kg−1	[36]
Hesperidin, narirutin, nobiletin, rutin, tangeretin	Peel, pulp	Ethanol	High-performance liquid chromatography	Hesperidin: 1685.6–2023.6 mg/100 g; Narirutin: 504.4–883.4 mg/100 g; Nobiletin: 131.2–185.2 mg/100 g; Rutin: 44.2–66.9 mg/100 g; Tangeretin: 21.3–26.3 mg/100 g	[37]
Hesperetin, nobiletin, tangeretin, quercetin, β-cryptoxanthin	Peel	Methanol	High-performance liquid chromatography, gas chromatography–mass spectrometry, ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometer	Hesperetin, nobiletin, tangeretin, quercetin: N/A; βCX: 680.93–1989.13 μg/100 g	[38]
Quercetin, rutin, naringin	Peel	Water	High-performance liquid chromatography	Quercetin, rutin: N/A; Naringin: 748 mg/L	[39]
Quercetin, hesperidin, hesperetin, narirutin	Peel	Methanol	High-performance liquid chromatography	Quercetin: 0.09–43.99 mg/g; Hesperidin: 76.81 mg/g; Hesperetin: 0.1 mg/g; Narirutin: 51.35 mg/g	[40]
β-cryptoxanthin	Pulp, juice	Ethanol	High-performance liquid chromatography	βCX: 0.8–5.5 μg/g−1	[41]
Synephrine	Juice	Water	High-performance liquid chromatography	Synephrine: 73.3–158 mg/L−1	[42]
Pectin	Peel	Ethanol	High hydrostatic pressure-assisted citric acid, hydrochloric acid	Pectin: 15.34–18.99%	[43]

N/A: not available.

. The chemical structure of these compounds is shown in

Table 2. Chemical structure of isolated compounds from Satsuma mandarin.

Classification	Chemical Structure	Ref.
Flavanones	Hesperidin	[44,45,46,47]
Flavanones	Hesperetin	[44,45,46,47]
Flavanones	Naringin	[45,46]
Flavanones	Naringenin	[44]
Flavanones	Narirutin	[44,46,47]
Flavanones	Neoponcirin	[48]
Polymethoxylated flavones	Nobiletin	[44,46]
Polymethoxylated flavones	Tangeretin	[44,46]
Polymethoxylated flavones	Heptamethoxyflavone	[49]
Alkaloids	Synephrine	[42]
Flavonols	Quercetin	[45,47]
Flavonols	Rutin	[45,47]
Flavonols	Quercetagetin	[50]
Carotenoids	β-cryptoxanthin	[51]
Dietary fibers	Pectin	[13]

. The flavanones were present in two forms: glycosylated (hesperidin, naringin, narirutin, neoponcirin) and aglyconated (hesperetin, naringenin). The immature peel of Satsuma mandarin was rich with hesperidin in areas located around the vascular bundles and between the albedo and flavedo layers [30]. Hesperidin, naringin, and narirutin were found in high amounts in the dry peels and/or pulps of various Satsuma mandarin cultivars [31]. Subcritical water extraction in combination with pulsed electric field resulted in enhanced extraction of narirutin and hesperidin from C. unshiu peels [32]. Using subcritical water extraction at temperatures ranging from 140 °C to 160 °C enhanced the yield of hesperidin and narirutin in C. unshiu peels [33]. Microwave-assisted extraction improved the overall recovery of narirutin and hesperidin from C. unshiu peels by 1310 mg/100 g and 5860 mg/100 g, respectively [34]. Hesperidin, narirutin, and naringin were the greatest amounts of flavanones found in the dry extract of C. unshiu peels made with 70% ethanol [35]. Higher levels of hesperidin, narirutin, and naringin were seen in Satsuma mandarins exposed to ozone during fruit storage after harvest [36].

Nobiletin, tangeretin, and 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF) were considered the major polymethoxylated flavones of Satsuma mandarin. Nobiletin and tangeretin levels are higher in the dry peels of five distinct Satsuma mandarin types than in the pulps and juices [31]. The peels of C. unshiu had higher levels of nobiletin and tangeretin than the pulps when the extracts were subjected to high temperatures (normal atmospheric temperature plus 4 °C), both during the day and at night [37]. The peels of C. unshiu were found to contain more nobiletin and tangeretin during the early stages of development than during the later stages of maturation [38].

The flavonols quercetin, rutin, and quercetagetin were also isolated from C. unshiu. Rutin extracted by 70% ethanol was the major compound present in C. unshiu peels [35]. Rutin and quercetin-3-O-rutinoside-7-O-glucoside were the major flavonols extracted from the juice of Satsuma mandarin [39]. Quercetin was found in the range of 0.09 to 43.99 mg/g among the phenolic components examined by high-performance liquid chromatography on the premature C. unshiu peel extract [40].

C. unshiu peels were found to have higher levels of βCX in both the early and late stages of citrus growth [38]. Mannitol and sucrose treatment enhanced the accumulation of βCX in C. unshiu’s juice sacs [41]. The synephrine level of C. unshiu juices collected from various groves was higher than that of orange juices [42]. High hydrostatic pressure-assisted citric acid produced greater quantities of pectin from C. unshiu peels than both hydrochloric acid and regular citric acid [43].

4. Pharmacological Activities of Satsuma Mandarin

In vitro/vivo experimental models have revealed that C. unshiu exerts anticancer, anti-obesity, anti-diabetic, cardioprotective, gastroprotective, neuroprotective, hepatoprotective, skin-protective, nasal airway-protective, lipid-lowering, antioxidant, anti-inflammatory, and anti-microbial activities. The results also showed that the main methods for identifying C. unshiu bioactives for pharmacological activity were solvent extraction (petroleum ether, ethanol, methanol, chloroform, water), freeze/hot-air-drying extraction, ultrasound-assisted alkaline extraction, and celluclast.

4.1. Anticancer

According to an experiment, the bioactives of C. unshiu (nobiletin, HMF, and tangeretin) inhibit 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine-induced cancer-associated mutations in the micronucleus test in vivo and the Ames test using the Salmonella typhimurium TA98 strain in vitro [52]. C. unshiu bioactives have shown therapeutic effects against a range of cancers through diverse activities and mechanisms of action. An experiment assessing the anticancer effect of C. unshiu ethanolic and petroleum ether extracts on the proliferation of melanoma skin cancer (MSC) cells showed that nobiletin, but not hesperidin, strongly suppressed the proliferation activity, with a half-maximal inhibitory concentration (IC₅₀) value of 35.65 µg/mL [53]. Another experiment evaluated the anti-metastatic effects of C. unshiu peel ethanolic extracts on MSC cells in vitro and in vivo. The extracts (hesperidin, naringin) demonstrated in vitro apoptosis induction in MSC cells through increasing the percentage of annexin V⁺/PI⁺/annexin V⁺/PI⁻ cells and the expression of Bcl-2-associated X protein (Bax), while decreasing the expression of B-cell lymphoma-2 (Bcl-2). The extracts also increased mitochondrial dysfunction in MSC cells via enhancing intracellular reactive oxygen species (ROS) formation, but decreased the migration, invasion, and colony formation of MSC cells through the inhibition of metalloproteinase (MMP) and the upregulation of tissue inhibitor of matrix metalloproteinases (TIMP) expression. In vivo, oral administration of C. unshiu peel extracts resulted in the inhibition of lung metastasis through reducing serum lactate dehydrogenase (LDH) activity and the expression of pro-inflammatory tumor necrosis factor-alpha (TNF-α) in B16F10 cell-inoculated mice [54].

An in vitro experiment evaluated the anti-metastatic activity of hesperidin and naringin extracted from C. unshiu peel in breast cancer (BRC) cells. The extracts inhibited invasion in cancer cells and adhesion to human umbilical vein endothelial cells induced by TNF-α by downregulating vascular cell adhesion molecule-1 (VCAM-1) expression and protein kinase C (PKC) phosphorylation [55]. Hesperidin and naringin extracted from C. unshiu peel suppressed BRC cell proliferation in vitro by activating apoptosis pathways, including cytosolic release of cytochrome c from mitochondria to cytoplasm, ROS-dependent AMP-activated protein kinase (AMPK) phosphorylation, as well as caspase-8/9, Poly(ADP-ribose) Polymerase (PARP), and Bax expression [56]. C. unshiu peel aqueous extracts triggered apoptosis and inhibited BRC cell viability at different concentrations in vitro through upregulating caspase-8/9, PARP, and Bax expression; promoting mitochondrial membrane potential loss/ROS production; and increasing cytochrome c release to cytoplasm from mitochondria [57].

Treatment with C. unshiu peel extracts (hesperetin, naringenin) inhibited pancreatic cancer (PaC) cell viability and migration in an in vitro and in vivo xenograft mouse model by downregulating the expression of proliferating cell nuclear antigen (PCNA) and upregulating the expression of cleavage caspase-3 through activating protein 38 (p38) and mitogen-activated protein kinase kinase 3/6 (MKK 3/6) signaling pathways [58]. Hesperetin combined with naringenin significantly induced apoptosis and suppressed PaC cell viability and migration when treated at different doses in both in vitro and in vivo xenograft mouse models through increasing caspase-3 cleavage and inhibiting the focal adhesion kinase (FAK) signaling pathway [59].

Oral administration of C. unshiu peel extracts (hesperidin, narirutin) to tumor-bearing mice inhibited tumor growth and proliferation in renal cell carcinoma (RCC) through decreasing the protein levels of TNF-α and increasing interferon-gamma (IFN-γ) expression [60]. C. unshiu and its extracts (hesperidin, narirutin) exerted in vitro antioxidant activity without causing cytotoxic effects in liver cancer cells treated with tert-butyl hydroperoxide-induced oxidative damage. The antioxidant activity is associated with decreased ROS production and glutathione (GSH) levels, and increased heme oxygenase-1 (HO-1) expression [61].

An experiment using a mouse model indicated that C. unshiu juice rich in hesperidin and βCX triggered apoptosis and suppressed proliferation in azoxymethane-induced colorectal cancer (CRC) cells by decreasing PCNA and cyclin D1 expression [62]. A diet supplemented with C. unshiu inhibited azoxymethane-induced CRC cell proliferation and decreased the number of colonic aberrant crypt foci, as well as β-catenin accumulated crypts in db/db mice [63]. Oral administration of C. unshiu aqueous extracts to CT-26 tumor-bearing mice markedly alleviated CRC-induced cachexia symptoms (weight loss, appetite, skeletal muscle atrophy) by decreasing pro-cachectic and inflammatory cytokine levels [64]. The aqueous extracts of C. unshiu have the potential to exert anti-cachectic, anti-inflammatory, and anti-tumor-growth activities following daily oral administration to CT-26 tumor-bearing mice and under treatment with doxorubicin, without causing toxic effects. The extracts significantly improved weight loss and reduced cachexia signs, as well as inflammatory cytokines, in tumor-bearing mice [65].

C. unshiu ethanolic extracts exerted inhibitory effects on the proliferation, viability, and colony formation of bladder cancer (BC) cells in vitro, which were associated with triggered apoptosis through modulating multiple gene expression and signaling pathways [66]. Treatment with the chloroform extracts of C. unshiu (hesperidin, heperetin) resulted in the suppression of proliferation, migration, and colony formation of cervical cancer (CC) cells in vitro. These effects were mediated by inducing apoptosis and cell cycle arrest at the G₂/M phase through altering different gene expression/signaling pathways [67].

The main findings related to the anticancer activities of C. unshiu bioactives are presented in

Table 3. Therapeutic activities of C. unshiu bioactives against different types of cancer.

Cancers	Model Type	Bioactive Compounds	Extraction Techniques and Application in Treatment	Anticancer Activity/Mechanisms	Target Genes/Signaling Pathways	Ref.
Melanoma	B16 mouse MSC cells	Hesperidin, nobiletin	B16 cells were treated with crude extracts from C. unshiu by petroleum ether and 95% ethanol at 15.63–250 µg/mL concentration for 72 h incubation at 70 °C. High-performance liquid chromatography was used to analyze the extracts.	Anti-proliferative	N/A	[53]
Melanoma	B16F10 mouse MSC cells, B16F10 cells-inoculated C57BL/6 mice	Hesperidin, naringin	B16F10 cells were treated with extracts from C. unshiu peel by 70% ethanol at different concentrations (0, 20, 40, 60, 80, and 100 µg/mL) for 24 h and incubated with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetra-zolium bromide solution for 2 h. The extracts were stored at −80 °C. High-performance liquid chromatography was conducted to identify the extracts. Mice were injected with phosphate-buffered saline and then randomized into 3 groups: B16 + control group (100 μL of distilled water), B16 + C. unshiu peel extracts 100 group (100 μL of C. unshiu peel extracts 100 mg/kg/day), and B16 + C. unshiu peel extracts 200 group (100 μL of C. unshiu peel extracts 200 mg/kg/day). Mice were orally administered for 21 days.	Apoptosis, anti-metastatic, anti-migration, anti-invasion, anti-inflammatory, inhibition of cell growth and colony formation	Bax, TIMP-1/2 ↑; Bcl-2, MMP-2/9, TNF-α, LDH ↓	[54]
Breast	MDA-MB-231 human BRC cells	Hesperidin, naringin	MDA-MB-231 cells were treated with C. unshiu extracts by 70% aqueous methanol and ethyl acetate elution at different concentrations (0, 10, 50, 100, and 200 µg/mL) for 24 h. The extracts were identified by high-performance liquid chromatography using a C18 column.	Anti-metastatic, anti-adhesion, anti-invasion	VCAM-1, PKC ↓	[55]
Breast	MCF-7 human BRC cells	Hesperidin, naringin	MCF-7 cells were treated with C. unshiu ethanolic extracts at different concentrations (0, 0.25, 0.5, 1, and 1.5 mg/mL) for 72 h. The extracts were pulverized into powder, boiled with water for 3 h, and then freeze-dried at −80 °C. The extracts were analyzed using high-performance liquid chromatography.	Anti-proliferative, apoptosis	caspase-8/9, Bax, acetyl-CoA carboxylase, PARP, ROS, AMPK, cytochrome c ↑; Bcl-2 ↓	[56]
Breast	MDA-MB-231 human BRC cells	NA	MDA-MB-231 cells were treated with dried C. unshiu peel water extracts at different concentrations (0, 0.25, 0.5, 1, and 1.5 mg/mL) for 72 h. The dried peel was pulverized into powder, boiled with water for 3 h, and then freeze-dried at −80 °C.	Anti-viability, apoptosis	caspase-8/9, Bax, PARP, cytochrome c, ROS ↑; Bcl-2, cIAP-1/2, XIAP, mitochondrial membrane potential ↓	[57]
Pancreatic	Panc-1 and SNU-213 PaC cells, BALB/c nude mice	Hesperidin, hesperetin, narirutin, naringenin	PaC cells were treated with C. unshiu peel extracts at different concentrations (0, 2.5, and 5 mg/mL) for 48 h. C. unshiu peel extracts were dissolved in sodium acetate buffer; incubated at 45 °C with β-glucosidase (90 U/g), cellulase (54 U/g), pectinase (120 U/g), and at 30 °C with fermivin. The quantification of C. unshiu peel extracts was conducted using limit of quantification and limit of detection values based on high-performance liquid chromatography. Mice were randomized into two groups to receive phosphate buffered saline or C. unshiu peel extracts (50 mg/kg).	Anti-viability, anti-migration, apoptosis	caspase-3, p38, MKK 3/6 ↑; PCNA ↓	[58]
Pancreatic	Panc-1, Detroit551, Miapaca-2, and SNU-213 PaC cells; BALB/c nude mice	Hesperetin, naringenin	PaC cells were treated with hesperetin and naringenin isolated from C. unshiu peel at different concentrations (0, 1, 5, 10, and 20 µM) for 48 and 72 h. The extraction was performed in sodium acetate buffer. The extracts were added to buffer and incubated with β-glucosidase (90 U/g), cellulase (54 U/g), pectinase (120 U/g) at 45 °C, and with fermivin at 30 °C. The extracts were analyzed using high-performance liquid chromatography. Mice were randomized to receive phosphate-buffered saline, 30 mg/kg hesperetin, 30 mg/kg naringenin, and 10/30 mg/kg naringenin/hesperetin mixture.	Anti-viability, anti-migration, apoptosis	caspase-3 ↑; FAK, p38 ↓	[59]
Renal	Balb/C mice renal cancer cell line Renca	Hesperidin, narirutin	Mice were supplemented with a standard diet and then randomized to receive phosphate-buffered saline plus water, or C. unshiu peel extracts (3 and 30 mg/kg) for 32 days, plus phosphate-buffered saline injection of Renca cells. The extracts were concentrated using a rotary evaporator and dried in a freeze-dryer, then dissolved in 400 mL of distilled water for 24 h at 4 °C and filtered. High-performance liquid chromatography–ultraviolet method was used for the detection of extracts.	Anti-proliferative, inhibition of tumor growth	IFN-γ ↑; TNF-α ↓	[60]
Liver	HepG2 human hepatic cells	Hesperidin, narirutin	HepG2 cells were treated with C. unshiu pulp juice extracts at different concentrations up to 200 µg/mL for 24 h. The pulp juice was filtered, concentrated using an evaporator at 60 °C, and then freeze-dried at −52 °C. High-performance liquid chromatography was used to detect the extracts.	Antioxidants	HO-1 ↑; ROS, GSH ↓	[61]
Colorectal	F344 rats	Hesperidin, β-cryptoxanthin	Rat were first injected with azoxymethane (20 mg/kg body weight) for 2 weeks to induce colonic neoplasms and then treated with a commercial C. unshiu juice rich in hesperidin and βCX (100 mg/kg) for 36 weeks. The centrifugation was applied to C. unshiu juice twice. The juice was pressed from the pulp and then frozen and thawed. This procedure was conducted again with adding 0.01% pectin to increase βCX levels.	Anti-proliferative, apoptosis	PCNA, cyclin D1 ↓	[62]
Colorectal	C57BL/KsJ-db/db mice	Hesperidin	Mice were injected with azoxymethane (15 mg/kg body weight) for 5 weeks and then supplemented with a diet containing C. unshiu segment membrane rich in hesperidin at concentration levels of 0.02%, 0.1%, and 0.5% for 7 weeks. Powdered C. unshiu consisted of 2.2 g hesperidin, 2.3 g ash, 5.5 g glucose, 6.1 g D-fructose, 15 g D-sucrose, 2.4 g moisture, 0.3 g fat, 5.5 g protein, 51 g fiber, and 9.7 g other flavonoids and unknown compounds.	Anti-proliferative	PCNA ↓	[63]
Colorectal	CT-26 mouse CRC cells, BALB/c mice	Hesperidin, naringin, narirutin, nobiletin	CRC cells were injected into the abdominal region of BALB/c mice. After tumor injection, mice were randomized to receive saline or C. unshiu aqueous extracts daily at concentrations of 250 and 500 mg/kg. The dried peel was dissolved in 1000 mL distilled water, extracted at 115 °C for 3 h, and then freeze-dried at 4 °C.	Anti-cachectic, anti-inflammatory	IL-6, TNF-α, IL-1β, MAFbx, MuRF-1 ↓	[64]
Colorectal	CT-26 mouse CRC cells, BALB/c mice	Hesperidin, naringin	CRC cells were injected with fetal bovine serum (10%), streptomycin (100 μg/mL), and penicillin (100 μg/mL) at 5% CO2. Mice were randomized to receive saline or C. unshiu peel aqueous extracts at a concentration of 350 mg/kg/day for 10 days. The peel was first dried and powdered. The extracts were steamed into the balloon, filtered, and stored at −20 °C.	Anti-cachectic, anti-inflammatory, anti-tumor growth	IL-6, TNF-α, IL-1β, MDA ↓	[65]
Bladder	T24 human BC cells	NA	BC cells were treated with C. unshiu peel ethanolic extracts at different concentrations (0, 100, 200, 400, 600, 800, and 1000 µg/mL) for 48 h. The dried peel was first powdered and extracted in 1 L of 70% ethanol for 24 h. The extracts were then concentrated using a vacuum rotary evaporator and freeze-dried at −80 °C.	Anti-proliferative, anti-viability, inhibition of colony formation, apoptosis	caspase-3/8/9, cytochrome c, Bax, PARP, ROS, TRAIL, FasL, tBid ↑; Bcl-2, Bid, XIAP, cIAP, PI3K, Akt ↓	[66]
Cervical	HeLa human CC cells	Hesperidin, heperetin	CC cells were treated with chloroform extracts of C. unshiu at different concentrations (0, 2, 4, 8, 16, 31, 63, 125, 250, and 500 µg/mL) for 72 h. The extracts were prepared using dimethyl sulfoxide at a concentration of 100 mg/mL. High-performance liquid chromatography analysis was conducted to detect hesperidin and heperetin contents.	Anti-proliferative, anti-migration, inhibition of colony formation, apoptosis, induction of cell cycle arrest	caspase-8/9, Bax, Bad, Fas ↑; Akt, PI3K, p21, p53, ERK1/2, cyclin B1/D1, Bcl-2, Bcl-XL ↓	[67]

(↓) Decrease; (↑) increase; NA = not available.

.

4.2. Anti-Obesity and Anti-Diabetic Activities

In experimental models, C. unshiu bioactives demonstrated an anti-obesity effect via various mechanisms. Oral administration of βCX from C. unshiu significantly reduced body weight, serum lipid levels, and adipocyte hypertrophy in experimental obese mice via upregulating and/or downregulating of genes involved in chemotaxis, cell proliferation, steroid metabolism, wound response, immune system development, lipid transport, fatty acid biosynthesis, muscle contraction, and DNA replication initiation [51]. The bioconversion of C. unshiu extracts with cytolase treatment promoted aglycoside forms (hesperetin and naringenin) and enhanced the anti-adipogenic activity in 3T3-L1 adipocyte cells through suppressing adipogenic transcription factors and inducing lipolytic activity [68]. Combined treatment with C. unshiu extracts (hesperidin) and Crataegus pinnatifida leaf effectively inhibited adipocyte differentiation in 3T3-L1 cells through downregulating the adipogenesis-related gene expression. The treatment also decreased the body weight, serum lipid levels, and visceral fat mass in Sprague–Dawley rats fed with a high-fat diet. These effects were mediated by multiple mechanisms, including activation of β-oxidation genes and downregulation of lipogenic and adipogenic genes [69]. Treatment with C. unshiu ethanolic extract and hesperidin induced the mRNA expression of uncoupling protein 3 (UCP3) in differentiated C2C12 myocytes. C. unshiu treatment also reduced the body fat contents and average fat cell size in skeletal muscle of obese mice fed with a high-fat diet by upregulating the mRNA expression of UCP3 [70]. Oral administration of celluclast extract from C. unshiu (narirutin) effectively reduced the body weight, white adipose tissue/liver weights, serum lipid levels, and hepatic lipid accumulation in high-fat-diet-induced obese mice through upregulating and/or downregulating of multiple genes/enzymes involved in lipogenesis, adipogenesis, and β-oxidation [71]. Treatment with Jeju roasted C. unshiu peel extracts resulted in the reduction of lipid accumulation during 3T3-L1 adipocyte differentiation. In obese mice fed with a high-fat diet, the treatment improved enzymes involved in lipid metabolism and decreased gene expression involved in lipid/energy metabolism [72].

In a few experimental rat models, C. unshiu also demonstrated anti-diabetic effects. Treatment with C. unshiu resulted in the reduction of hyperglycemia-induced oxidative stress in Streptozotocin-induced diabetic rats liver. This was mediated by decreasing serum liver enzyme levels and improving antioxidant enzyme levels [73]. Goto-Kakizaki rats demonstrated reduced blood glucose levels and improved glucose tolerance after 10 weeks feeding of C. unshiu [74]. The administration of C. unshiu to type 2 diabetic db/db mice significantly reduced weight gain, hepatic phosphoenolpyruvate carboxykinase enzyme, hepatic fat accumulation, hepatic cholesterol, free fatty acid and triglyceride contents, plasma glucose levels, glucagon, triglyceride, and free fatty acid through decreasing hepatic lipid-regulating enzymes and modulating hepatic inflammatory genes [75].

Table 4. Therapeutic activities of C. unshiu bioactives against obesity and diabetes.

Model Type	Bioactive Compounds	Extraction Techniques and Application in Treatment	Therapeutic Activities/Mechanisms	Target Genes/Signaling Pathways	Ref.
Obese mouse model	β-cryptoxanthin	Mice were assigned to two groups: tsumura suzuki obese diabetes group (experiment) and tsumura suzuki non-obese diabetes group (control). The experimental groups were fed with enzyme-processed Satsuma mandarin suspended in olive oil (400 mg/kg/day; 0.8 mg of βCX/kg/day). The control groups were supplemented with olive oil only.	Reduced adipocyte hypertrophy, body weight, cell proliferation, inflammatory chemokines, and excess immune responses in adipose tissue	Steroid metabolism genes: Hmgcs1, Cyp51, Idi1 ↑; Hdlbp, Abca1 ↓ DNA replication initiation genes: Mcm 2/4/5/6 ↓ Chemotaxis, cell cycle, and immune system development genes: Cdk1, Ccna2, Ccnb1, Ccnb2, Cxcl2/10, Ccl2/3/4/7/12 ↓ Inflammatory/anti-inflammatory genes: adiponectin ↑; TNF-α, MCP-1 ↓ Lipid transport, wound response, fatty acid biosynthesis, and muscle contraction genes: Myh2/7, Tpm3, Tnnc1 ↑; Elovl6, Scd1, Fasn, ApoA 1/2, ApoC 1/3, Fga, Kng, Serpinc1 ↓	[51]
3T3-L1 adipocyte cells	Hesperetin, naringenin,	3T3-L1 cells were treated with C. unshiu, C. unshiu with cytolase, and Sinetrol at a concentration of 0.5 mg/mL for 24 h during 10-day adipocyte differentiation. Extraction of 100 g dried C. unshiu peel was conducted with 190 units/g cytolase PCL5 in 2 L of d-H2O by incubation for 14 h at 60 °C. The peel was enzymatically treated and filtered using a filter paper, which was then extracted in 8 L of 80% ethanol for 8 h at 60 °C. High-performance liquid chromatography was used to analyze C. unshiu peel extracts.	Anti-adipogenic, lipolytic	PPARγ, C/EBPα, SREBP1c ↓	[68]
3T3-L1 adipocyte cells, Sprague–Dawley rats	Hesperidin	3T3-L1 cells were treated with HT048 (C. unshiu peel extracts and Crataegus pinnatifida leaf) at different concentrations (0, 50, 100, 200, 400, and 800 μg/mL). HT048 was extracted in 30% ethanol at 90 °C for 4 h, filtered, and freeze-dried to produce a dark-yellow powder. HT048 extracts were analyzed using high-performance liquid chromatography. Rats were randomized to receive a chow diet, high-fat diet, high-fat diet with orlistat, and high-fat diet supplemented with HT048 (0.2%, 0.4%) for 12 weeks.	Reduced adipocyte differentiation, body and fat weight, and serum lipid levels; promoted glycerol release	β-oxidation genes: PPARα, CPT-1 ↑ Adipogenic genes: PPARγ, C/EBPα mRNA ↓ Lipogenic genes: SREBP1c, FAS mRNA ↓	[69]
C2C12 mouse myocytes, C57BL/6 obese mice	Hesperidin	C2C12 cells were treated with C. unshiu at different concentrations (10, 50, and 100 μg/mL) and hesperidin (35 μg/mL) for 24 h. The dried C. unshiu peel was extracted in 4 L of 70% aqueous ethanol at 80 °C for 1 h. Mice were randomized to receive a normal diet (AIN-76A) for 10 weeks, high-fat diet, and high-fat diet (60% calories from fat) supplemented with C. unshiu extracts (75 mg/kg/day).	Reduced fat mass/body weight and average fat cell size	UCP3 ↑	[70]
C57BL/6 obese mice	Narirutin	Mice were randomized to receive a high-fat diet (containing 60% kcal from fat), low-dose high-fat diet supplemented with C. unshiu extracts (125 mg/kg/bw), and high-dose high-fat diet supplemented with C. unshiu extracts (200 mg/kg/bw) once daily for 11 weeks. A total of 10 g of dried C. unshiu powder was first mixed with 100 μL of Celluclast enzyme and 1 L of distilled water, and then incubated for 24 h at 50 °C.	Inhibited adipogenesis, lipogenesis, and hepatic lipid accumulation; reduced serum lipid levels	Lipogenic genes/enzymes: SREBP1c, FAS ↓ Adipogenic genes/enzymes: PPARγ, C/EBPα ↓ β-oxidation genes/enzymes: AMPK, ACC ↑	[71]
3T3-L1 adipocyte cells, C57BL/6 obese mice	NA	3T3-L1 cells were treated with Jeju roasted peel extract at concentrations range from 50 to 200 μg/mL. Mice were randomized to receive a normal diet, high-fat diet (containing 60% kcal from fat), and high-fat diet supplemented with Jeju roasted peel extract (25/50 mg/kg/bw).	Reduced lipid accumulation in adipocytes	Enzymes: ALT, ASAT, γ-GTP ↑ Lipogenic genes: SREBP1c, FAS ↓ Adipogenic genes: PPARγ, C/EBPα ↓	[72]
Wistar rats	NA	Rats were first fed with a standard diet (Oriental yeast) for 10 weeks and injected with Streptozotocin (75 mg/kg) dissolved in a citrate buffer. Rats were then assigned to three groups: 0%, 1%, or 3% (wt/wt) C. unshiu-treated diabetic groups.	Inhibited hyperglycemia-induced liver dysfunction	Antioxidant enzymes: SOD, GSSG, GSH ↑ Liver enzymes: ALT, γ-GTP ↓	[73]
Goto-Kakizaki rat model of type 2 diabetes	NA	Rats were fed with 1% and 3% (wt/wt) C. unshiu for 10 weeks.	Reduced glucose/non-fasting blood glucose levels; improved glucose tolerance	NA	[74]
C57BL/KsJ-db/db type 2 diabetic mice	Hesperidin, narirutin	Mice were supplemented with rosiglitazone (0.001 g/100 g diet) or C. unshiu peel ethanol extract (2 g/100 g diet) for 6 weeks. The extraction of 50 g dried C. unshiu peel was performed with 1 kg of 60% ethanol by incubation at 70 °C for 3 h. The extracts were then cooled, filtered, and freeze-dried at −40 °C.	Reduced blood glucose levels, body fat mass, body weight gain, plasma lipid levels, hypertriglyceridemia, and hepatic steatosis	Hepatic lipid regulating enzymes: FAS, ME, PAP, HMGR ↓ Hepatic inflammatory genes: adiponectin and IL-10 ↑; IL-6, TNF-α, IFN-γ, MCP-1↓	[75]

(↓) Decrease; (↑) increase; NA = not available.

presents the anti-obesity and anti-diabetic activities of C. unshiu bioactive compounds.

4.3. Hepatoprotective and Lipid-Lowering Activities

Few studies have examined the hepatoprotective and lipid-lowering activities of C. unshiu bioactives in vitro and in vivo. An experiment was designed to evaluate the protective effect of C. unshiu peel extracts against non-alcoholic fatty liver disease in mouse hepatocyte cells (AML12) and high-fat-diet-fed rats. Cells were treated with C. unshiu extracted through hot-air-drying (50 °C for 72 h) at different concentrations (25, 50, and 100 µg/mL). High-performance liquid chromatography was used to analyze C. unshiu extracts. Rats were randomized into different groups to receive double-distilled water, a 60% high-fat diet, a 60% high-fat diet with double-distilled water and C. unshiu extracts (50/100 mg/kg), and a 60% high-fat diet with metformin (100 mg/kg). Hesperidin, the major extract of C. unshiu, activated AMPK phosphorylation and regulated palmitate-induced hepatocyte steatosis in AML12 cells by inhibiting the mammalian target of rapamycin complex 1 (mTORC1)-endoplasmic reticulum (ER) stress pathway. Administration of C. unshiu and hesperidin also reduced hepatic fat accumulation in high-fat-diet-fed rats by decreasing triglycerides levels, total cholesterol, lipid peroxidation, alanine aminotransferase (ALT), and aspartate aminotransferase (ASAT) through activating the AMPK pathway and suppressing the mTORC1-ER stress response associated with ROS accumulation [76]. Another experiment evaluated the hepatoprotective effects of Diospyros kaki and C. unshiu mixture on nonalcoholic fatty liver disease in high-fat-diet-fed rats. Boiling water was used to extract 500 g of dried C. unshiu peel. Rats were orally administered a 45% high-fat diet for 23 weeks (control group) and C. unshiu extracts at concentrations of 50 or 100 mg/kg for two months (experimental group). Compared with the control group, the administration of C. unshiu extracts improved lipid metabolism and reduced liver damage in high-fat-diet-fed rats by activating peroxisome proliferator-activated receptor alpha (PPARα), uncoupling protein 2 (UCP2) expression, and AMPK phosphorylation, while inhibiting the expression of fatty acid synthase (FAS), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), acetyl-CoA carboxylase (ACC), stearoyl-CoA desaturase-1 (SCD-1), and sterol regulatory element binding protein-1 (SREBP-1) [77]. An in vivo experiment aimed to investigate the hepatoprotective and bone-protective activities of dried C. unshiu peel extracts in ovariectomized rats. The dried peel (300 g) was extracted with 70% ethanol (3 L) at 80 °C for 3 h. The extracts were then filtered, concentrated, and freeze-dried to produce a dark-yellow powder. Rats were assigned to the control group (ovariectomized plus 10 μg/kg/day 17β-estradiol) and experimental group (ovariectomized plus dried C. unshiu peel extracts at concentrations of 30, 100, and 300 mg/kg/day). Hepatic lipid contents/accumulation, serum lipoprotein levels, and femur bone mineral density loss were reduced in rats fed C. unshiu extracts (mainly hesperidin) [78].

The hypolipidemic and bifidogenic activities of dietary fiber containing arabinose, xylose, and galactose prepared from the dried albedo of Mikan (C. unshiu) were assessed using an experimental mouse model. The albedo was milled and freeze-dried to yield a fine powder. WistarHan GALAS rats were first fed with a standard diet for two days, and then randomized into the control group (received a diet containing 5% cellulose) and experimental group (received a diet containing 4% cellulose and 1% total dietary fiber extracted from C. unshiu for 4 weeks). Serum triglyceride levels and triacylglycerol/cholesterol contents in the liver were decreased in the experimental group compared to control groups. This decrease was mainly due to the suppression of pancreatic lipase and increased caecal bifidobacteria counts [79].

4.4. Gastroprotective Activity

The bioactives of C. unshiu have been shown in experimental models to have strong gastroprotective activities. In a mouse model of ulcerative colitis, the therapeutic effects of C. unshiu water extracts (hesperidin and narirutin) were investigated. The dried peel (300 g) was extracted with water at 100 °C for 2 h using a hot water extractor. The extracts were then evaporated at 50 °C, freeze-dried, and stored at −80 °C. The experiment was performed on male Balb/c mice induced by acute colitis. Ulcerative colitis mice were randomized into control, distilled water-treated, 100 mg/kg sulfasalazine-treated, and 100/200 mg/kg C. unshiu peel water extract-treated groups. Compared to other groups, the sulfasalazine and C. unshiu peel water extract-treated groups showed a significant reduction in weight gain, colon length, and inflammatory cell infiltration/inflammatory lesions. These effects were mediated by the decrease in oxidative stress biomarkers (ROS; malondialdehyde, MDA; NADPH oxidase 2, NOX2), inflammatory cytokines (interleukin IL-6/IL-1β; cyclooxygenase-2, COX-2; inducible nitric oxide synthase, iNOS), and apoptotic-related proteins (Bax, caspase-3), as well as the inhibition of phosphatidylinositol 3-kinase/threonine kinase (PI3K/Akt) and mitogen-activated protein kinase/nuclear factor kappa B (MAPK/NF-κB) signaling pathways [80]. The bioconversion of C. unshiu peel containing naringenin and hesperetin was evaluated in Caco-2 epithelial cells and murine with dextran sodium sulfate-induced colitis. C. unshiu peel was extracted with 70% ethanol at room temperature for 24 h. Caco-2 cells were treated with C. unshiu extracts at a dose of 50 μg/mL. Female C57BL/6 mice were orally administered C. unshiu extracts at a dose of 500 mg/kg for two weeks and then treated with 3% dextran sodium sulfate for one week. C. unshiu extracts reduced gut inflammation, improved the intestinal permeability of Caco-2 cells by increasing the mRNA levels of occludin, and inhibited the activation of canonical NF-κB through decreasing the expression of IkappaB kinase (IκB) and protein 50 (p50). The administration of C. unshiu to mice demonstrated anti-inflammatory effects by decreasing the frequency of T-helper 17 (Th17) cells in the large intestine [81].

An experiment assessed the efficacy of pectin extracted from C. unshiu using male C57BL/KsJ-db/db and db/m mice by regulating the gut microbiota and its metabolites. Mice were first given access to food and water for one week and then assigned to db/m mice as the control group, type 2 diabetes model group, metformin-positive group (100 mg/kg), pectin from basic/acid water group (200 mg/kg), and commercial pectin group (200 mg/kg). Mice were orally administered pectin for four weeks. Pectin administration not only decreased the fasting blood glucose/glycated serum protein and serum lipid profiles, but also modulated the gut microbiota composition in mice by increasing the relative abundance of specific microbiota (Lactobacillus, Ligilactobacillus, Limosilactobacillus) and promoting the production of metabolite short-chain fatty acids (SCFAs) [82]. An experimental mouse model was used to study the effects of dietary fiber extracted from C. unshiu on intestinal microecology and SCFAs contents. Mice were first injected with d D-galactose solution (200 mg/kg/d) for 42 days and then orally administered soluble dietary fiber (low/high doses 100/200 mg/kg/d) for 28 days. The treatment with soluble dietary fiber reduced MDA, and increased SOD and glutathione peroxidase activity (GSH-Px) in the liver/serum of D-galactose-stimulated oxidative stress mice. Mice receiving high doses of soluble dietary fiber exhibited high microbial richness and butyric acid concentration, while those receiving low doses demonstrated high levels of valeric acid, acetic acid, and butyric acid. The treatment also enhanced the equilibrium of intestinal flora through a positive correlation between isovaleric acid and Firmicutes, and a negative correlation between acetic acid and Muribaculaceae, as well as caproic acid and Lachnospiraceae_NK4A136_group [83]. The ultrasound-assisted alkaline extraction of soluble dietary fiber from C. unshiu exhibited free radical scavenging capacity, enhanced the levels of propionic acid, and increased the abundance of Bacteroides [84].

An experimental model was conducted by investigating the effects of dried mature/immature C. unshiu aqueous extracts (hesperidin) on gastrointestinal motility in rodents. The dried peel (2 kg) was extracted with distilled water at 100 °C for 3 h. The extract solution was then filtered, concentrated, and lyophilized. Male Sprague–Dawley rats and Swiss albino mice were first given access to a standard diet and tap water for one week and then treated with dried mature/immature C. unshiu peel at different doses (0, 0.25, 0.5, 1, 2, or 5 g/kg) for two weeks. The treatment with dried mature C. unshiu extracts significantly accelerated the intestinal transit rate in rodents, with no clinical signs of toxicity at 5 g/kg or below observed [85]. Another in vivo experiment investigated the effects of dried mature C. unshiu peel and hesperidin on gastric mucosa. The peel was extracted with 700 mL water and refluxed for 90 h at 60 °C. The extracts were then vacuum-filtered and lyophilized. Wistar rats were randomized into the control group, aspirin administration group, aspirin plus C. unshiu administration group (20 mg/kg BW), and aspirin plus hesperidin administration group (20 mg/kg BW). The results showed that C. unshiu and hesperidin significantly inhibited oxidative DNA damage and gastric mucosal injury induced by aspirin through reducing the content of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) [86]. The effects of C. unshiu peel ethanolic extracts on gastrointestinal tract motility were assessed in the interstitial cells of Cajal derived from BALB/c mice small intestine. The dried peel (600 g) was extracted with 70% ethanol (6 L) at 20 °C for 2 h. Cells were exposed to muscarinic receptors (M2/M3 receptor antagonist; 4-1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; methoctramine), the active phospholipase C inhibitor U-73122, and MAPK inhibitors (p38 MAPK inhibitor, p42/44 MAPK inhibitor, SB203580, PD98059, c-jun NH2-terminal kinase JNK inhibitor) at a dose of 10 μM before treatment with 5 mg/mL C. unshiu extracts. Results showed that C. unshiu-induced membrane depolarization was suppressed following treatment with M3 muscarinic receptor antagonist, the active phospholipase C inhibitor U-73122, and MAPK inhibitors (SB203580, PD98059) [87].

4.5. Neuroprotective Activities

A few experimental investigations have shown that different mechanisms mediated the neuroprotective action of C. unshiu bioactives. According to these experiments, mice treated with C. unshiu extracts demonstrated reduced peripheral neuropathy, oxidative stress in astrocytes, and neurotoxicity in the hippocampus and cerebral cortex. The neuroprotective activity of C. unshiu methanolic extracts (nobiletin, HMF, and tangeretin) was evaluated in astrocytes (glial cells) and Mongolian gerbils. Immature C. unshiu powder (10 g) was suspended in methanol (50 mL) at room temperature. The extracts were then evaporated and suspended in distilled water. Cells were first preincubated with C. unshiu extracts for 30 min and then treated with lipopolysaccharide (LPS) at a dose of 500 ng/mL for 24 h. Gerbils were fed with a commercial powder or 1% C. unshiu and water ad libitum. Treatment with C. unshiu extracts suppressed LPS-induced nitric oxide (NO) and iNOS expression in astrocytes through downregulating the NF-κB and p38-MAPK signaling pathways. Oral administration of C. unshiu extracts to gerbils delayed ischemia/reperfusion in the hippocampal CAI region [88]. Swiss albino mice and human neuroblastoma SH-SY5Y cells were used to test the antidepressant activities of an ethanolic extract of C. unshiu dried peel. The peel (600 g) was extracted with 70% ethanol (6 L) at 80 °C for 6 h. Cells were treated with C. unshiu extracts (hesperidin and narirutin) at different concentrations (10, 50, or 100 μg/mL) and a synthetic glucocorticoid dexamethasone at a concentration of 200 μM for 24 h. Mice were first injected with 40 mg/kg dexamethasone and then orally administered 30, 100, and 300 mg/kg C. unshiu extracts for two weeks. In SH-SY5Y cells, the extracts demonstrated reduced neurotoxicity and depressive-like behaviors induced by dexamethasone. The extracts also ameliorated dexamethasone-induced neurotoxicity in the cerebral cortex and hippocampus of mice by upregulating the expression of tropomyosin receptor kinase B (TrkB), brain-derived neurotrophic factor (BDNF) level, and cyclic AMP-response element-binding protein (CREB) [89]. The aim of an experiment was to determine the antioxidant activity of C. unshiu ethanolic extracts in Neuro2A cells and preclinical mice. Cells were treated with C. unshiu extracts to test the inhibition of hydrogen peroxide (H₂O₂). Mice were first treated with oxaliplatin-based chemotherapy to induce peripheral neuropathy and then orally administered C. unshiu extracts for four weeks. Treatment with C. unshiu extracts significantly inhibited H₂O₂ in Neuro2A cells and decreased the expression of iNOS- and NOX2-mediated oxidative stress in mice with peripheral neuropathy [90].

4.6. Skin-Protective Activity

Bioactives from C. unshiu shown skin-protective activities through a variety of mechanisms. In vitro experiment showed that fermented C. unshiu peel extracts (hesperetin and naringenin) with the Schizophyllum commune QG143 strain significantly increased the biosynthesis of collagen in human dermal fibroblasts exposed to ultraviolet and decreased photoaging skin through a mechanism that involves the reduction of MMP-1 mRNA expression and β-galactosidase (SA-β-gal) levels [91]. Treatment with HMF extracted from C. unshiu peel suppressed collagenase activity and increased type I procollagen synthesis in vitro via upregulation and/or downregulation of protein/gene expression in the ultraviolet-induced human dermal fibroblast neonatal cells [92].

An experiment was performed using human keratinocyte cells and the atopic dermatitis mouse model to investigate the effects of premature C. unshiu ethanolic extracts on atopic dermatitis. C. unshiu treatment led to a significant decrease in the mRNA/protein expression of inflammatory chemokines in TNF-α- and IFN-γ-induced keratinocyte cells through suppression of signal transducer and activator of transcription 1 (STAT1) phosphorylation. In dinitrochlorobenzene (DNCB)-treated mice, C. unshiu significantly reduced atopic dermatitis symptoms (e.g., redness, wrinkles) in the dorsal skin and the production of T-helper-type (Th1/Th2) cytokines [93]. An experiment on the anti-inflammatory effects of immature C. unshiu ethanolic extracts on atopic dermatitis demonstrated that quercetagetin caused an inhibitory effect on the protein/mRNA expression of thymus- and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) produced in keratinocyte cells through the downregulation of the Janus kinase (JAK)/STAT signaling pathway [50]. Fermentation of C. unshiu byproducts with nuruk (a starter inoculated with airborne microorganisms) for 15 days reduced oxidative stress induced by H₂O₂ in human keratinocyte cells [94]. The ethanolic extracts of fermented dried C. unshiu using Bacillus subtilis showed skin moisturizing efficacy in keratinocyte cells through stimulating the formation of hyaluronic acid and the expression of serine palmitoyltransferase (SPT) and filaggrin. The extracts also exerted anti-inflammatory activity in murine macrophages by decreasing the release of LPS-stimulated NO, iNOS, COX-2, TNF-α, IL-6, and prostaglandin 2 (PGE₂) [95].

In experimental Swiss albino mice, the combination of C. unshiu or hesperidin and prednisolone effectively suppressed ear swelling during the effector/induction phase of contact dermatitis induced by picryl chloride [96]. The ethanolic extracts of C. unshiu showed a reduction in oxidative stress and an inhibition of tyrosinase enzyme responsible for melanin synthesis in melena cells, without inducing any toxic effect. Oral administration of C. unshiu extracts to guinea pigs also demonstrated a reduction of ultraviolet-induced skin pigmentation [97]. Oral administration of C. unshiu powder to ultraviolet B-irradiated hairless mice dorsal skin led to a significant increase in skin moisture content and a decrease in transepidermal water loss, basement membrane degradation, and epidermal hyperplasia [98].

Experimental studies that have evaluated the skin-protective activity of C. unshiu bioactives are summarized in

Table 5. Therapeutic applications of C. unshiu bioactives in skin conditions.

Model Type	Bioactive Compounds	Extraction Techniques and Application in Treatment	Therapeutic Activities/Mechanisms	Target Genes/Signaling Pathways	Ref.
Human dermal fibroblasts	Hesperetin, naringenin	Dermal fibroblasts were treated with the aqueous extracts of C. unshiu peel at different concentrations (0.025–0.1%, w/v) for 24h in the submerged culture of Schizophyllum commune QG143 strain. C. unshiu peel (100 g) was extracted with distilled water (1 L) at 85 °C and then filtered and evaporated to produce a raw residue. High-performance liquid chromatography analysis was carried out to identify the extracts.	Increased the biosynthesis of collagen from fibroblast; decreased photoaging skin	MMP-1, SA-β-gal ↓	[91]
Human dermal fibroblast neonatal cells	Heptamethoxyflavone	Cells were treated with dried C. unshiu peel ethanolic extracts at different concentrations (50, 100, 200, and 400 µg/mL) for 24h. The peel (20 g) was extracted with 50% ethanol (5 L) at room temperature for 7 days. The extracts were evaporated and mixed with hexane/water (1 L) to yield the hexane-soluble fraction.	Decreased photoaging skin; increased type I procollagen; inhibited collagenase activity	Type I procollagen protein, Smad3 ↑; MMP-1, Smad7, MAPK, ERK, JNK, c-Fos ↓	[92]
HaCaT human keratinocyte cells, atopic dermatitis mouse model	NA	Cells were first induced with TNF-α and IFN-γ (10 ng/mL) in the presence or absence of premature C. unshiu ethanolic extracts at different concentrations (25, 50, 100, and 200 μg/mL) and then treated with 20 μL 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide for 4 h. The powder (37.95 kg) was extracted with 80% ethanol (5 L) at 60 °C for 4 h. The extracts were evaporated for 3 h, filtered, and freeze-dried for 60 h Mice first received a standard diet and ad libitum water and then assigned to four groups (induction, positive control, normal, and C. unshiu). Mice in the dinitrochlorobenzene (DNCB) group (induction) were sensitized by applying 100 μL of 1% DNCB in acetone and 100 μL of 0.5% DNCB to the dorsal skin for 36 days. Premature C. unshiu extracts were administrated every 2 days from day 16. Mice in the positive control group received HYDCORT cream (2 mg/g hydrocortisone valerate).	Anti-atopic, anti-inflammatory	TNF-α, IFN-γ, IL-4, TARC/CCL17, MDC/CCL22, STAT1 ↓	[93]
HaCaT human keratinocyte cells	Quercetagetin	Cells were first stimulated with TNF-α and IFN-γ (10 ng/mL) for 24 h in the presence or absence of immature C. unshiu ethanolic extracts and other flavonoids at different concentrations (12.5, 25, and 50 μM) and then treated with 10 μL 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide for 4 h. High-performance liquid chromatography analysis was used to identify quercetagetin.	Anti-atopic; reduced inflammatory response	TNF-α, IFN-γ, IL-4, TARC/CCL17, MDC/CCL22, JAK, STAT1 ↓	[50]
HaCaT human keratinocyte cells	Hesperetin, naringenin, quercetin	Cells were treated with fermented C. unshiu byproduct by 70% ethanol (25 °C for 24 h) at a concentration of 100 μg/mL, followed by induction with H2O2 at a dose of 500 μM.	Protected against oxidative stress	H2O2 ↓	[94]
HaCaT human keratinocyte cells, RAW264.7 murine macrophages	Hesperidin, hesperetin, nobiletin, tangeretin, narirutin, narigenin, rutin	Cells were pretreated with the isolated Bacillus subtilis strains WE(1-4) and AL(2-1), which were added to dried C. unshiu peel ethanolic extracts at a concentration of 10% (v/v) for 96 h RAW264.7 macrophages were pretreated with WE(1-4) and AL(2-1) for 2 h and then stimulated with 1 μg/mL LPS for 22 h.	Reduced inflammatory response; moisturizing effect	Hyaluronic acid, SPT, filaggrin ↑; NO, iNOS, COX-2, TNF-α, IL-6, PGE2 ↓	[95]
Swiss albino mice	Hesperidin	Mice were first sensitized by a topical administration of 0.1 mL picryl chloride solution (7%) in ethanol to the shaved abdomen. C. unshiu extracts or hesperidin were suspended in 0.2% carboxymethylcellulose sodium and administered orally at a dose of 0.2 mL/10 g from day 1 to 5 over 7 days. C. unshiu extracts or hesperidin in combination with prednisolone (0.1 mL/10 g) were then administrated for 7 days.	Inhibited ear swelling of contact dermatitis induced by picryl chloride	NA	[96]
Melana melanocytes, brown guinea pigs	Hesperidin, heptamethoxyflavone, β-cryptoxanthin	Cells were treated with C. unshiu ethanolic extracts (70%) at different concentrations (1, 5, 10, and 50 μg/mL) or vitamin C (50 μg/mL) for 72 h. Pigs were exposed to ultraviolet B radiation (380 mJ/cm2) three times per week over two weeks, and then randomized into groups to receive water (control group) and C. unshiu extracts (50 and 250 mg/kg).	Inhibited melanin synthesis; reduced oxidative stress and skin pigmentation	Tyrosinase enzyme, ROS ↓	[97]
Hos:HR-1 hairless mice	Hesperidin, narirutin	Mice were given access to a laboratory diet and water, and then assigned to ultraviolet non-irradiated/irradiated control mice and C. unshiu groups (200 mg/kg for seven weeks).	Improved photoaging skin	NA	[98]

(↓) Decrease; (↑) increase; NA = not available.

.

4.7. Cardioprotective Activity

According to some experiments, the bioactives in C. unshiu exhibit cardioprotective activities. An experiment was designed to assess the proangiogenic effects of C. unshiu aqueous extracts (hesperidin and narirutin) in human umbilical vein endothelial cells. Cells were cultured in plates and starved for 15 h in endothelial cell growth basal medium-2 prior to C. unshiu extracts stimulation at concentrations of 2.5 or 5 mg/mL. Cell proliferation, migration, and tube formation were markedly decreased upon treatment with C. unshiu extracts. This effect may have been caused by the activation of tyrosine-phosphorylated proteins, as well as the phosphorylation of FAK and extracellular signal-regulated kinase 1/2 (ERK 1/2) [99]. Another experiment focused on the rat aortic ring model, as a tool to assess the effects of C. unshiu ethanolic extracts on vasoconstriction. C. unshiu peel was extracted with 70% ethanol at 70–80 °C for 3 h. The extracts were then filtered, concentrated, and lyophilized at −20 °C. The thoracic aorta from rats was isolated and divided into ring segments of 4–5 mm length. Nobiletin and synephrine extracts from C. unshiu were subsequently applied to the ring segments at different concentrations (5, 10, 15, and 100 µm) for 30 min. This resulted in a notable suppression of serotonin/phenylephrine-stimulated vasoconstriction [100].

4.8. Nasal Airway-Protective Activity

The activity of C. unshiu and its extracts to protect the nasal airways has been demonstrated in a few experiments. The objective of an experiment was to investigate the anti-allergic activity of C. unshiu methanolic extracts in basophils of patients with allergic rhinitis and rat basophilic leukemia RBL-2H3 cells. Basophils from leukocyte fractions arranged by dextran sedimentation were suspended in 100 μL of Tyrode buffer solution. Cedar pollen allergen extract (0.1 μg/mL) was then added to basophils, and the effects of adding C. unshiu extracts (1.6 mg/mL) or wortmannin (25 μM) were examined. RBL-2H3 cells were washed with modified Tyrode buffer, wortmannin (25 μM), or C. unshiu extracts (1.6, 8, or 16 mg/mL). Citrus hesperetin and nobiletin reduced histamine release from basophils in patients and β-hexosaminidase release from RBL-2H3 cells through the inhibition of Akt-1 phosphorylation and the PI3K signaling pathway [101]. In an experiment using an animal model, rats were orally administered unripe mandarin extracts (hesperidin, narirutin) using subcritical water, leading to a significant improvement in the peripheral blood flow after allergic sensitization with hen egg white lysozyme. Oral administration of extracts also inhibited nasal airway resistance in ovalbumin-challenged guinea pigs [102].

4.9. Antioxidant Activity

Numerous experiments have thoroughly assessed the antioxidant activity of C. unshiu bioactives. In one experiment, the antioxidant activity of sunburned and non-sunburned fruits of C. unshiu was evaluated. The peel and pulp of C. unshiu were dried at 45 °C for two days, ground into powder, and then extracted by adding 30 mL of 70% ethanol to the powder. The extracts (hesperidin, narirutin, and naringin) exerted significant antioxidant activity in moderate–severe sunburned C. unshiu compared to those in the non-sunburned (control). Sunburned fruits showed lower IC₅₀ values for diammonium salt (ABTS⁺) radical scavenging activity and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity than non-sunburned fruits, indicating a high level of antioxidant activity in C. unshiu [35]. The antioxidant activity of flavanones from C. unshiu exposed to ozone treatment was determined using the DPPH and ABTS⁺ methods. C. unshiu peel was frozen in liquid nitrogen, powdered, stored at −80 °C, and then extracted with 70% methanol (10 mL). The DPPH activity of C. unshiu peel extracts (hesperidin, narirutin, and naringin) was increased following exposure to ozone on day 1 (8.7%) and day 4 (8.5%). On the other hand, the ABTS⁺ activity was increased on day 1 and day 4 by 4.2% and 2.1%, respectively [36]. The methanolic extracts of C. unshiu peel during different stages of development were investigated for antioxidant activity. After harvesting C. unshiu fruits on different dates in 2018, the peel was removed, freeze-dried, powdered, and stored at −80 °C. The highest IC₅₀ values for the extracts (hesperetin, nobiletin, tangeretin, quercetin, and βCX) were observed in harvested C. unshiu on Nov. 16 using the DPPH radical assay (8192.77 mg/mL) and the ABTS⁺ radical assay (1181.56 mg/mL) [38]. The antioxidant activity of premature and mature C. unshiu peel extracts were investigated in vitro. Premature and mature C. unshiu peel were first dried at 80 °C for 9 h, ground with a blender into a 40-mesh size, and extracted with methanol (1:10 w/v) at 50 °C for 24 h. The extracts hesperidin and narirutin showed high antioxidant activity evaluated with five methods: H₂O₂ scavenging activity, SOD activity, DPPH activity, ABTS⁺ activity, and hydroxyl radical scavenging activity. Hesperidin and narirutin demonstrated higher hydroxyl radical and H₂O₂ scavenging activities (74–98%) than the other tests [40].

Semi-continuous subcritical water extraction was used to investigate the antioxidant activity of bioactive components from the peel of C. unshiu. The peel was first dried for three days, ground into powder, stored at 20 °C, and then extracted with pure methanol for 30 min. Among active compounds, hesperetin exerted the highest DPPH activity (115.8 ± 3.9 mg AAE/g), oxygen radical absorbance capacity (ORAC) (710.9 ± 12.0 mg TE/100 mg), and ferric-reducing antioxidant power (FRAP) (549.1 ± 21.1 mmol FSE 100/g) [44]. The antioxidant activity of C. unshiu peel extracts in LPS-stimulated RAW 264.7 macrophages was assessed in vitro. The peel was ground into powder (100 g), lyophilized at 80 °C, extracted with 70% aqueous methanol for 24 h, purified, and then evaporated at 40 °C. Three compounds with highly antioxidant activity were isolated from C. unshiu peel and identified as hesperidin, naringin, and rutin. These compounds significantly decreased ROS production and increased antioxidant enzymes (SOD, catalase) in macrophages [45]. Identification of bioactive compounds from C. unshiu flowers and assessment of their DPPH activity were the objectives of an experiment. After extracting 195.0 g of extract from 3.2 kg of fresh C. unshiu flowers using methanol, the pure components were separated using column chromatography over silica gel, ODS, and Sephadex LH20. Among the isolated compounds, quercetin and rutin demonstrated the highest radical scavenging activities against DPPH, with IC₅₀ values of 11.5 and 19.6 μM, respectively [47].

Several techniques were used to evaluate the antioxidant activity of quercetagetin from the peel of C. unshiu. The dried peel was first powdered and extracted in 800 mL L⁻¹ methanol for 12 h. The extracts were then evaporated and concentrated with chloroform (10.06 g), n-butanol (25.58 g), and ethyl acetate (14.55 g). The ethyl acetate fraction (6.5 g) was purified by elution with ethyl acetate/methanol (10:1–1:1 v/v) and n-hexane/ethyl acetate (10:1–1:1 v/v) to produce 15 fractions. High-performance liquid chromatography was performed to separate fraction 11.5.5 (2.3 mg) with 400 mL L⁻¹ methanol to produce the final compound (0.8 mg). Quercetagetin showed high DPPH activity (IC₅₀ = 7.89 μmol L⁻¹) and reduced ROS formation in Vero cells, as well as H₂O₂-induced DNA damage [103]. Using the Trolox equivalent antioxidant capacity (TEAC), the antioxidant activity of the C. unshiu peel extracts produced by ultrasound treatment has been assessed. After extracting the dried C. unshiu peel using 20 mL of diethyl ether/ethyl acetate (1:1, v/v), 80% methanol was added to bring the final amount down to 10 mL. The methanolic solution was filtered using a 0.45 μm microporous membrane and the filtrate analyzed by high-performance liquid chromatography. Ultrasound treatment at 15 or 30 °C for 20 min resulted in enhanced extraction of hesperidin and narirutin. Following treatment, TEAC and total phenolic acid levels showed a positive association (R2 = 0.8288 at 15 °C, R2 = 0.7706 at 30 °C), indicating that these phenolic acids may have a part in boosting the antioxidant activity of C. unshiu peel [104].

4.10. Anti-Inflammatory Activity

Some experiments have assessed the anti-inflammatory activity of C. unshiu bioactives. The anti-inflammatory activity of the aqueous and methanol peel extracts of C. unshiu (hesperidin, naringin, and rutin) were investigated in LPS-stimulated RAW 264.7 macrophages. The extracts exhibited anti-inflammatory activity by reducing the release of LPS-stimulated PGE₂, NO, iNOS, COX-2, TNF-α, and IL-1β in macrophages [45]. The inhibitory effects of C. unshiu peel on the production of inflammatory cytokines/mediators were investigated in LPS-induced RAW 264.7 cells. The dried peel was dissolved in 1000 mL distilled water, extracted at 115 °C for 3 h, and then freeze-dried at 4 °C. The active compounds hesperidin and naringin were identified in C. unshiu peel by high-performance liquid chromatography. These compounds significantly inhibited NO, PGE₂, IL-6, TNF-α production, iNOS and COX-2 expression, as well as p38, ERK, JNK, MAPK, and NF-κB phosphorylation, in macrophage cells [105]. The anti-inflammatory effects of dried immature C. unshiu fruit extracts by 70% ethanol and ethyl acetate elution were evaluated in LPS-stimulated RAW 264.7 cells. The extracts exhibited anti-inflammatory activity by decreasing iNOS and COX-2 protein expression through the suppression of MAPK and NF-κB signaling pathways [106]. An experiment evaluated the anti-inflammatory effects of C. unshiu aqueous extracts rich in hesperidin using RAW 264.7 cells/mouse peritoneal macrophages in vitro and LPS-induced systemic inflammation mice. RAW 264.7 cells and mouse peritoneal macrophages were treated with C. unshiu at different concentrations (101, 102, 103, 104 µg/mL) for 24 h, followed by LPS induction for 6 h. Mice were assigned to control, LPS, and LPS plus C. unshiu groups. The LPS plus C. unshiu group was orally administered C. unshiu extracts at a dose of 300 mg/kg for 7 days, while the other groups received water only. Treatment with C. unshiu extracts inhibited LPS-induced NO, iNOS, TNF-α, IL-6, IL-1β, and CCL2 expression in RAW 264.7 cells and mouse peritoneal macrophages through downregulating the JNK, NF-κB, and p38-MAPK signaling pathways. The treatment of C. unshiu raised the expression of the anti-inflammatory cytokine IL-10 while decreasing the expression of TNF-α and IL-6 in comparison to other groups [107].

4.11. Anti-Microbial Activity

The anti-microbial activity of C. unshiu aqueous methanol peel extracts was investigated against Penicillium digitatum (P. digitatum). The isolated flavanones and flavones (e.g., hesperidin, nobiletin, tangeretin) were identified by high-performance liquid chromatography using a C18 column. After infection, the levels of active compounds in C. unshiu first declined before progressively rising again. The stimulation of the flavanones/flavones biosynthesis pathway to acquire resistance to P. digitatum may be the cause of this alteration [46].

4.12. Summary of Pharmacological Activities

Overall, this review’s findings demonstrated that the bioactives of C. unshiu, both in vitro and in vivo, exhibit anticancer, anti-obesity, anti-diabetic, cardioprotective, gastroprotective, neuroprotective, hepatoprotective, skin-protective, nasal airway-protective, lipid-lowering, antioxidant, anti-inflammatory, and anti-microbial activities. An assessment of the bioactive compounds and their pharmacological actions in C. unshiu is shown in

Table 6. Summary of C. unshiu Marc’s bioactive compounds and pharmacological activities.

Pharmacological Activities	Bioactive Compounds
	Hesperidin, hesperetin, nobiletin, naringin, naringenin, narirutin, β-cryptoxanthin
	Hesperidin, hesperetin, naringenin, narirutin, β-cryptoxanthin
	Hesperidin, narirutin
	Hesperidin, dietary fiber
	Hesperidin, dietary fiber
	Hesperidin, hesperetin, naringenin, narirutin, dietary fiber, pectin
	Hesperidin, narirutin, nobiletin, heptamethoxyflavone, tangeretin
	Hesperidin, hesperetin, nobiletin, naringin, naringenin, narirutin, quercetin, quercetagetin, rutin, heptamethoxyflavone, tangeretin, β-cryptoxanthin
	Hesperidin, narirutin
	Hesperetin, nobiletin
	Hesperidin, nobiletin, naringin, narirutin, quercetin, quercetagetin, rutin, tangeretin, β-cryptoxanthin
	Hesperetin, naringin
	Hesperidin, nobiletin, tangeretin

.

5. Conclusions

Bioactive compounds found in Satsuma mandarin are abundant and showed a range of therapeutic benefits in both in vitro and in vivo studies. The main analytical techniques used to identify C. unshiu extracts for pharmacological activity were petroleum ether, ethanol, methanol, chloroform, water, freeze/hot-air-drying, celluclast, and ultrasound-assisted alkaline.

Hesperidin, hesperetin, nobiletin, narirutin, naringin, and βCX are among the bioactives from C. unshiu that demonstrated anticancer activities against cells from the breast, pancreas, kidneys, liver, colorectal, bladder, cervical, and melanoma. The anticancer activities were attributed to a number of pathways, including the induction of cell cycle arrest/apoptosis and the prevention of cell growth, colony formation, adhesion, migration, invasion, proliferation, metastasis, and inflammation. Extracts from C. unshiu, including hesperidin, hesperetin, narirutin, naringenin, and βCX, demonstrated anti-obesity and anti-diabetic activities by lowering blood glucose levels, hypertriglyceridemia, hepatic steatosis, adipocyte hypertrophy/differentiation, fat mass/body weight, serum lipid levels, and hyperglycemia-induced liver dysfunction. The hepatoprotective and lipid-lowering mechanisms of C. unshiu bioactives may be linked to their capacity to control palmitate-induced hepatocyte steatosis and lower hepatic fat accumulation, lipid peroxidation, liver damage, serum lipoprotein levels, triglyceride levels, and total cholesterol.

Hesperetin, narirutin, and narirutin demonstrated gastroprotective activity through enhancing intestinal permeability, modifying gut microbiota, speeding intestinal transit rate, decreasing gut inflammation, and preventing mucosal damage and oxidative harm to DNA. Evaluations have been conducted on the gastroprotective activity of C. unshiu bioactives and other traditional medications. In mice treated with sulfasalazine and C. unshiu bioactives, inflammatory lesions were significantly reduced. Mice administered aspirin plus C. unshiu showed a substantial reduction in both aspirin-induced gastric mucosal damage and oxidative DNA damage. Pectin treatment increased the relative abundance of certain bacteria, which in turn altered the composition of the gut microbiota in mice when compared to metformin treatment.

Animal models showed neuroprotective activity from the interaction of C. unshiu bioactives. The combination of nobiletin, HMF, and tangeretin, for instance, inhibited the production of iNOS and NO in astrocytes (glial cells) caused by LPS and delayed ischemia/reperfusion in the hippocampus CAI area in Mongolian gerbils. In the mice’s hippocampal and cerebral cortex, narirutin and hesperidin both reduced the neurotoxicity caused by dexamethasone.

The skin-protective activity of C. unshiu bioactives (hesperetin, hesperetin, naringenin, HMF, nobiletin, tangeretin, neoponcirin, narirutin, quercetagetin, quercetin, and rutin) increased collagen biosynthesis from fibroblasts, as well as decreased melanin synthesis, skin inflammation/pigmentation, oxidative damage, and photoaging skin. C. unshiu nobiletin and synephrine demonstrated cardioprotective activity by inhibiting serotonin and phenylephrine from inducing vasoconstriction. Histamine/β-hexosaminidase secretion and nasal airway resistance were reduced by the main bioactives of C. unshiu, hesperetin, nobiletin, narirutin, and hesperidin, which demonstrated nasal airway-protective activity.

The antioxidant activity of C. unshiu bioactives has been proven by experiments using a variety of techniques, such as the DPPH, ABTS⁺, FRAP, hydroxyl radical, and H₂O₂ scavenging activities. Through several mechanisms, the anti-inflammatory activity of C. unshiu bioactives has been demonstrated in LPS-induced RAW 264.7 cells and the animal model, resulting in the reduction of the production of inflammatory cytokines and mediators. Bioactives from C. unshiu also demonstrated anti-microbial activity by contributing significantly to resistance against P. digitatum.

6. Limitations and Future Perspectives

The utilization of C. unshiu bioactives in clinical studies was not assessed, despite the fact that they demonstrated a broad spectrum of therapeutic activities when examined in vitro and in vivo. The majority of in vivo studies were carried out on mouse models; however, some were carried out on guinea pigs. The bioactives of C. unshiu have been shown in a limited number of studies to have non-toxic effects. The pharmacological actions of C. unshiu bioactives were not determined by any tests that showed their bioavailability. The effective dose for attaining the best therapy effect was unclear, despite the fact that each experiment assessing the bioactives of C. unshiu demonstrated therapeutic efficiency.

More experiments are still needed in order to clarify the mechanisms of action of C. unshiu bioactives and investigate their therapeutic potential. Additional investigations on the possible toxicity at various doses are required to ascertain the applicability of C. unshiu bioactives. Further experiments are required to ascertain the best dosage and mode of administration for C. unshiu extracts. Clinical studies are required to evaluate the therapeutic activities of C. unshiu bioactives. Preclinical animal investigations are needed to ascertain the therapeutic potential of C. unshiu bioactives before being translated to clinical practice.