Review of the nutritional composition , medicinal , phytochemical and pharmacological properties of Citrus reticulata Blanco ( Rutaceae ) [ version 1 ; peer review : 1 approved ]

Citrus reticulata Blanco is a moderately-sized fruit tree widely used as herbal medicine worldwide. The nutritional composition, medicinal uses, phytochemistry and pharmacological properties of C. reticulata were critically reviewed in the current study. The literature linked to C. reticulata properties was obtained from multiple internet sources including Elsevier, Google Scholar, SciFinder, Web of Science, Pubmed, BMC, Science Direct, and Scopus. Ethnopharmacological research identified antioxidants such as vitamin C, carotenoids and phenolic compounds, also a source of sugars, organic acids, amino acids, pectins, minerals and volatile organic compounds as components of C. reticulata. As a medicinal plant, C. reticulata is used for the treatment of dyspepsia, gastro-intestinal distension, cough with profuse phlegm, hiccup and vomiting. The crude extracts of C. reticulata fruits have depicted anti-inflammatory, anticholesterolemic, analgesic, antiasthmatic, antiscorbutic, antiseptic, antitussive, carminative, expectorant, stomachic. With more people becoming nutritionconscious, there has been an increase in the demand for the use of citrus fruits and their by-products as traditional medicines for conventional healthcare in developing countries.


Introduction
Citrus reticulata Blanco is a large species belonging to the family Rutaceae, with various varieties and hybrids 1 . It includes popular citrus types such as Satsumas, Clementines, Tangerines 2 and the Mediterranean mandarin 3,4 . Tangerine is a group of orange-coloured citrus fruits consisting of mandarin hybrids 5 , although the term tangerine is used interchangeably with mandarin.
Mandarins, like other citrus species, are indigenous to the subtropical and tropical zones of Asia, particularly China and Cochin-China 6,7 . Some researchers have reported that mandarins, alongside other citrus species, evolved in a region including Vietnam, South China, India and Japan 8,9 . They are now widely cultivated around the world in the warm temperate and tropical areas 8,10-12 . Mandarins account for 22-25 per cent of world citrus production among the commercially cultivated citrus species 9,13,14 . The major citrus growing regions of the world and their estimated output is shown in Figure 1.
Mandarins, including both monoembryonic and polyembryonic cultivars and many interspecific hybrids, are the most diversified category of citrus fruits 15,16 . Nevertheless, a remarkable similarity has been documented between mandarin cultivars at molecular and isoenzymatic levels [17][18][19] . There are 36 mandarin species, according to Tanaka 20 , while Swingle recognized only three species, one of them being C. reticulata consisted of 34 species of Tanaka's system 21 . Chromosome studies have shown that the genus has a stable chromosome number of 2n=18, except for a few polyploids, with a small number of chromosome markers in the conventional karyotype 22 . The medicinal uses, phytochemistry and pharmacological properties of C. reticulata were examined in the current report.

Botanical description
The mandarin plant is a spiny, evergreen, bushy shrub growing 2-8 m tall 23 with most varieties averaging 7.5 m 13 . The tree has a dense top with slender branches bearing dark green, lanceshaped leaves with a prominent midrib. Petioles are narrowly winged or slightly margined. This tree bears white scented flowers followed by oval to flattened, sweet-fleshed, golden fruits 13 . The mandarin fruit resembles other oranges, is smaller and more oblate than oranges, round in shape, orange in colour, sweet in taste, with a thin, loose, easy-to-peel skin and can be easily damaged by cold. The fruit is up to 8 cm in diameter with easily separable segments 2 .
Flowering is induced through low-temperature stress or soil water deficit stress 24,25 . Under subtropical climates, flowering is an annual event occurring during spring [26][27][28] . In tropical areas, flowering is a continuous event, mostly determined by moisture availability from sufficient rain or water supply 27-29 while in temperate regions it occurs with the onset of winter 30 .

Methods and justification of the study
The literature search was performed from March 2020 to June 2020. A mixed-method review approach which involved combining quantitative and qualitative research was used to compile the review. Information on nutritional composition, medicinal uses, phytochemical and pharmacological properties of C. reticulata was gathered from textbooks, theses and online research articles from databases such as Elsevier, Google Scholar, Scopus, Science Direct, SciFinder, PubMed, BMC, and Web of Science. These data sources were chosen based on the topic covered and the main search key terms included "taxonomy, botany, distribution, nutritional composition, ethnobotanical uses, biological and chemical properties" in relation to C. reticulata. Search terms were set to be in the title, keywords and abstract. To avoid too much filtering of literature, the terms were searched individually. Focusing, on its multipurpose roles, C. reticulata production and utilization can be a catalyst for the development of rural households and community livelihoods. It is therefore imperative to document its nutritional composition, medicinal use and pharmacological properties. With more people becoming nutrition-conscious, demand for citrus fruits and their by-products has grown even in developing countries.

Nutritional composition
Mandarin is a rich source of vitamins C and A, proteins, dietary fibres and essential minerals such as calcium, potassium, phosphorus and magnesium. Also, they contain minute quantities of vitamins B1, B2, B3, B5, B6, B9 and E 8,13 . On average, 100 g of mandarin orange consists of 85% water (85.2 g), 13% carbohydrates (13.34 g), 0.81 g protein, 0.38 g dietary fibre and 0.31 g fat 8,13 . Table 1 shows the nutritional composition of raw mandarins.
The sugars, acids, carotenoids, polyphenols, limonoids and vitamins in C. reticulata determine the flavour of the fruit. The vitamins, fibre and health-boosting plant compounds like flavonoids provide many health benefits to humans who eat the fruits and related by-products. For example, the vitamin B complex helps prevent infections, helps promote cell health, energy levels, proper nerve functions, hormone and cholesterol production, and cardiovascular health. In particular, mandarin fruits are rich in β-cryptoxanthine, xanthophyll with pro-vitamin A activity 31 .
Mandarins are well-accepted by consumers owing to their pleasant flavours and abundant phytochemicals. With more people becoming nutrition-conscious, there has been a growing demand for citrus fruits like mandarins, and their by-products 13 .

Chemical constituents
The chemical constituents of C. reticulata peel, juice and fruit are shown in Table 2. The peel has high magnesium and carotenoid content 32 . Methyl-N-methyl anthranilate, a natural antinociceptive compound, has been isolated from mandarin leaves 33 . Secondary metabolites such as terpenoids, flavonoids and phenolics compounds act as deterrents to insects and microbial attack 34 .

Essence oil and aroma
Mandarin is a source of essential oils which are characterized by a fresh-juice fragrance that is widely used in citrus juice products as a natural flavoring agent 8,35 . The essential oils contain volatile compounds, mainly aldehydes, limonene, ketones, esters, alcohols, terpenes, β-myrcene, 3-carene and α-pinene which provides the distinctive aromas and tastes of citrus fruits 1,36,37 . Limonene, preceded by γ-terpinene, p-cymene, alpha-pinene and myrcene, is the most abundant compound in mandarin essential oil 1,38-43 .
The essential oils are greatly utilized as fragrance materials in beverages, foods, medical formulations, perfumery, toiletries and other cosmetic products 44 . To some extent, they can also be used as traditional medicine 2,9,45 . The Chinese use the dried peel of the fruit in the regulation of ch'I (energy/vitality) and to enhance digestion. The leaves and juvenile twigs are a source of essential oil called 'petitgrain oil' 23 .

Medicinal uses
The edible part of the raw mandarin fruit possesses antioxidants such as vitamin C, carotenoids and phenolic compounds. The fruit is also a rich source of amino acids, sugars, organic acids, amino acids, pectins, minerals and volatile organic compounds 46-51 . These constituents are essential for the proper functioning of the body by protecting it against chronic diseases and providing basic nutrition 52 . The dietary fibre and phenolic compounds in mandarins are useful in the formulation of functional foods 32 . Mandarin fruit also contains coumarins, for instance, bergapten which sensitizes the skin to sunlight 53 .
The fruit has been reported to possess laxative, aphrodisiac, antiemetic, astringent and tonic properties 54,55 while the fruit peel regulates skin moisture, softens hard and rough skin and cleanses oily skin 56 . Traditionally, it is also used as a stomachic and carminative [56][57][58] . Both the pericarp and endocarp are anticholesterolemic, analgesic, antiseptic, antiasthmatic, anti-inflammatory, antiscorbutic, antitussive, carminative, expectorant and stomachic 57,67 . Therefore, they are used in the treatment and management of dyspepsia, gastro-intestinal distension, cough with profuse phlegm, hiccup and vomiting 58,67 . The unripened green exocarp is used in the treatment of chest pains and hypochondrium, gastro-intestinal distension, swelling of the liver and spleen and cirrhosis of the liver. The seed is analgesic and carminative, thus used in the treatment of hernia, lumbago, mastitis and pain or swellings of the testes 67 .
Pharmacological properties of C. reticulata Antimicrobial properties The ethanolic extract of mandarin fruit shell, a traditional herbal medicine used for gastric ulcer treatments in China, showed activity against five clinical strains of Helicobacter pylori at the minimum inhibitory concentration (MIC) close to 60 µg/mL 68  Sultana et al. 57 have reported that the volatile oil of C. reticulata peel possesses antimicrobial activities against Escherichia coli, Staphylococcus aureus, Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus, and Candida albicans. Thus, suggested that the volatile oil could be useful for the treatment of skin disorders and the therapy can be incorporated into the cosmetic formulation. Based on the findings of this review, the essential oil, juice and peel extracts of C. reticulata may possess beneficial antibacterial agents that can be exploited in controlling unwanted bacterial infections. The peel oils of mandarins exhibit toxic insecticidal and antibacterial properties 8 .

Anticancer activity
Kang et al. 73 have reported that the methanol extracts (100 g/mL) of C. reticulata peel showed increased apoptosis on SNU-C4, human colon cancer cells through Bax-related caspase-3 activation, thus, suggested the use of C. reticulata on colon cancer patients. In an in vitro study, two flavone glucosides isolated from the mandarin fruit peel showed differentiation-inducing activity in mouse myeloid leukaemia cells (M1), and the cells exhibited phagocytic activity 74 . In addition, hexane and dichloromethane bark extracts of C. reticulata assayed against human lung adenocarcinoma cell line A549, human breast adenocarcinoma cell line MCF7, human Caucasian prostate adenocarcinoma cell line PC3, and one normal human prostate cell line PNT2 revealed that the extracts possess good apoptosis-inducing activity against the human cancer cell lines. Thus, the authors concluded that the hexane or dichloromethane extract of the bark of C. reticulata is a good crude drug treatment against lung, breast and prostate cancer. However, further in vitro and in vivo testing would be required before any recommendation of this drug can be given 75 .
The anticancer potential of Citrus medica (2 morphotypes), C. sinensis, C. maxima, C. limon and C. reticulata peels were investigated using in vitro assays and in vivo cancer models 76 . The finding depicted that both the extracts and EOs of C. reticulata peels had significant activity against Dalton's Lymphoma Ascites (DLA) cell line in an MTT assay. The peel oil showed 91.9% and 100% cell death at 25 and 50 µg/mL, respectively, while the water extract showed 49.8% cell death at 5 µg/mL and 100% cell death at 25 and 50 µg/mL respectively. The in vivo study revealed that mice pre-treated with C. reticulata peel extract were significantly (50%) protected from DLA compared to post-treated mice (33%) without any obvious toxic symptoms. The volatiles (essential oils, limonoids) and nonvolatiles (mainly polymethoxy flavones) in Citrus peels have been recognized as their bioactive/anticancer constituents 76,77 .

Neuropharmacological activities
Gbaj et al. 78 evaluated the anxiolytic potentials of methanol and aqueous peels extracts of C. reticulata in Libya using an elevated plus-maze. The result revealed that the peel extracts exhibited significant anxiolytic activity. In addition, the anxiolytic effect of naringin has been confirmed in 6-8 weeks old mice weighing 30 to 35 g 79 .

Antigenotoxicity effects
Hassan et al. 64 investigate the protective effect of the ethanol extract of the aerial part of C. reticulata cultivated in Saudi Arabia against genotoxicity induced by benzo(a)pyrene (BaP) in mice using the comet assay. In the mice treated with BaP, there was a significant increase in the DNA fragmentation in the liver tissues of male mice and an increased rate of DNA damage in mice blood cells. However, the liver and blood cells of the mice treated with ethanol extract demonstrated significant protection by inhibiting the rate of DNA damage. It was concluded that the aerial part of C. reticulata could be useful to reduce the genotoxicity induced by hazardous chemical agents 64 . The presence of flavonoid compounds and various secondary metabolites could be responsible for the protective effect, pharmacological and therapeutic properties of C. reticulata [80][81][82] .

Antioxidant effects
Boudries et al. 43 investigated the antioxidant activities of C. reticulata, C. reticulata cultivar Wilking and C. clementine from Algeria using 1,1-diphenyl-2-picrylhydrazil (DPPH) and reducing power. The essential oil (EO) of C. reticulata exhibited the strongest DPPH free radical-scavenging activity in a dose-dependent manner, followed by clementine and wilking EOs. Also, in a concentration-dependent manner, the EO of C. reticulata showed the greatest reducing power followed by wilking and clementine EOs. According to Junior et al. 83 , the antioxidant nature of the citrus essential oils in terms of free radical scavenging may be due to the antioxidant activity of limonene, which was the main constituent of the oil. The peel of C. reticulata was evaluated for antioxidant activity, the results displayed prominent, concentration-dependent free-radical scavenging activity on stable DPPH free radicals and reactive hydroxyl radicals 84 .
Also, the fruit peel of C. reticulata, Zingiber officinale and Sesamum indicum were investigated for their antioxidant activities using the DPPH radical scavenging technique. The findings revealed that Z. officinale had the highest antioxidant activity followed by C. reticulata, and S. indicum. The antioxidant activity of these plants could be attributed to a wide variety of constituents, such as the flavonoid content which are considered as major biological antioxidants 85 .

Cardiovascular effects
Rincon et al. 32 suggested the use of tangerine peel in reducing the risk of cardiovascular diseases and some associated with lipid oxidation.

Hepatoprotective activity
The protective effect of the essential oils of C. reticulata on isoniazid induced hepatotoxicity in Wistar rats was investigated 86 . About 50 gm/kg, p.o. of isoniazid was administered for 30 days in order to induced liver damage in the rats. A total of 200 mg/kg, p.o.of the essential oil was administered daily for 30 days, while the standard group received Liv52 86 . The result revealed a significantly elevated level of ALT, AST, bilirubin and a decreased total protein content in the rats treated with only isoniazid as compared to the group that do not received isoniazid. However, a significant reduction in all the biochemical parameters was observed in the rats treated with the essential oil and Liv52 86 .

CYP450 effects
In an in vitro study, the effect of tangeretin (a flavonoid isolated from tangerine juice) on hydroxylation of midazolam, a CYP3A4 probe was evaluated using human liver microsomes and recombinant CYP3A4. The finding revealed that tangeretin is a potent and regioselective stimulator of midazolam 1'hydroxylation and complementary DNA-expressed CYP3A4 87 . However, further studies are required as the authors have indicated that tangerine juice might not have a clinical effect on CYP3A-mediated drug metabolism in humans.

Antihypercholesterolemic effects
Omer et al. 65 investigated the antihypercholesterolemic potential of the crude ethanolic extracts of C. reticulata fruit peel in an in vivo study. The findings revealed that daily administration of 250 mg/kg and 500 mg/kg doses of the extracts to the albino rats for four weeks produced a reduction in serum low-density lipoprotein-cholesterol, total cholesterol and triglycerides levels. Also, a significant elevation in serum high-density lipoproteincholesterol was observed, thus, indicating their cardioprotective effects and potential as therapeutic antihypercholesterolemic agents 65 . Hence, the efficacy of C. reticulata peels extracts on the observed lipid profile parameters might be attributed to the presence of polymethoxylated flavones which occur in the fruit peels.
Anti-ageing potential According to a study by Apraj and Pandita 66 , both hot and cold alcoholic extracts of C. reticulata exhibited strong anti-collagenase and anti-elastase activity, indicating its anti-ageing ability. However, further study is required to determine whether the extracts can be incorporated into skincare products as anti-wrinkle agents.

Toxicity
The oral administration of C. reticulata extracts up to 200 mg/kg has been reported to be safe 78 . Also, Li et al. 68 reported that a single oral dose of 16 g/kg of naringin does not produce acute oral toxicity in rats.

Other uses
Worldwide, the mandarin fruit is eaten as fresh produce 88 . It is peeled and eaten plain, used in salads, desserts and main dishes or cooked in puddings, cakes and confectionery. The peel, pulp and seeds are often discarded as waste or they can be processed into animal feed 88 .
Many by-products, including pectin, dried pulp, molasses, marmalades, candied peel, peel seasoning, purees, beverage bases, alcohol, bland syrup, citric acid, seed oil and flavonoids can be obtained from mandarin fruits 4,36,89 . The dried peel or rind has a sweet-spicy flavour often used as a flavouring in cakes 4 or as a spice for cooking, baking, drinks or candy 90 . Rind powder extract, a rich source of phenolic compounds having free radical scavenging activity can be used as an anti-oxidant in meat products 88 .

Conclusion
C. reticulata is an important plant which contains some compounds and nutritional values that are of great health importance. The fruit is rich in antioxidants and phenolic compounds, sugars, organic acids, amino acids, pectins, minerals and volatile organic compounds. These substances are essential for the proper functioning of the body by protecting it against chronic diseases, provides basic nutrition and useful in the formulation of functional foods. Based on research carried out, the fruits and peels of C. reticulata have been reported to possess neuropharmacological, hepatoprotective, anticancer, antimicrobial, antigenotoxicity, antioxidant activities, antihypercholesterolemic and cardiovascular effects.  Reviewer Expertise: Plant science on agronomy, biochemistry and phytochemisty I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.