Introduction
Citrus fruits are the best sources of ascorbic acid (vitamin C) and secondary metabolitessuch as phenolic acid, coumarins, carotenoids, flavonoids, alkaloids, limonoids. They are rich in B vitamins, folate, essential minerals, and other bio-active compounds, also. So, the citrus genus is considered one of the best anti-inflammatory, antioxidant, anti-cancer, and neuroprotective agents. In recent days, various citrus fruits are considered for extensive research to determine their effects on complementary medicine and chemotherapy applications. Six Citrus species have already been included in the respective pharmacopeia of many countries, mainly China, Japan, Korea, Indonesia, Nepal, Bhutan, and India.1
Properties of citrus fruits
The citrus fruits are popular due to their sensory attributes:taste, color, and astringency. Hence, their organoleptic properties altogether attract many consumers apart from theirantioxidant and immuno-protective roles. In general, they can help incough and cold, indigestion, ringworm infections, controlling blood pressure, skin and hair health, and managing other inflammations, as well.2, 3
Sweet lime (Citrus limetta)
This review study focused only on sweet lime (Citrus limetta), known as 'Mousambi' or 'Mosambi' in India.Its components are exclusively used for multiple clinical applications considering their various therapeutic benefits not only in Asian countries but also in the western world. It is regarded asone of the Indian superfoods due to its pharmacological effects.Traditionally,sweet lime has been usedto prevent scurvy, skin and hair issues, GI intolerance, constipation, type-II diabetes, ulcers, urinary tract infections, and overall, boosting innate immunity.Earlier studies have shownthat there are many effective compounds present in Sweet limepulp, areresponsible for its antioxidant and anti-inflammatory functions. d-limonene is one of these bioactive compounds (BAC)available in sweet lime. It is the principal constituent of Citrus limettathat offers pharmacokinetic and pharmacodynamics properties. Phytochemical analyses show that there are so many essential phytoconstituents like tannins,alkaloids, flavonoids, glycosides, anthraquinones, phenolic, terpenes, saponins, essential oils present in Citrus limetta; around 30 bioactive compounds are identified with the help of mass spectrophotometry, which are available in fruit pulp, juice even in the peel, as per various studies. The main component, limonene in the citrus peel oils, constitutes about 45%-94.6% in concentration out of the total fruit portion.4, 5, 6
Production of sweet lime
Among all the continents, Europe did the highest business on lemons and limes during 2019, with the shipmentvalue at $1.5 billion, i.e., 42% of the total in the world market. India and china contribute majorly to lime production worldwide. Andhra Pradesh (≈1800 tonnes), Maharashtra (≈500 tonnes),and Telangana (≈300 tonnes) are the top three sweet lime-producing states in India, sustainable forthe last five years in India.7
The graphical presentation of the production of Mosambi in India has been showing in Figure 1. India is gradually progressing the production of this fruit due to its high demand.
Figure 1
Production of Sweet Lime based on top 10 states in India since 2014 till 2020. (financial year).
Source: National Horticulture Board (NHB)-Government of India; https://agriexchange.apeda.gov.in/
Materials and Methods
This review studyis primarily focused to review and analyzethe inhibitory and complementary therapeutic effects of sweet lime's pulp and juice with respect to the RNA virus-neutralizing properties, mainly of SARS-CoV-2.This study was designed and based on rigorous online searches of relevant open-access literature available in best quality and reliable databases such asthe National Center for Biotechnology Information (NCBI), mainly Pubmed, Biomed (BMC), Directory of Open Access Journals (DOAJ),and Google Scholar.Keywords and phrases used for online searches: 'virucidal effect of sweet lime (Citrus limetta),' 'role of mosambi on RNA viruses,' 'therapeutic action of sweet lime,' inhibitory effect of sweet lime.' Other relevant keywords were also applied along with boolean operators OR, AND, NOT.8, 9, 10
Discussions
Nutrients and Bioactive components
Sweet lime (Citrus limetta) as a citrus fruit is rich in vitamin C and other minerals and plenty of other adequate nutrients that all together providean inhibitory, complementary therapeutic effect. As per the Indian Food Composition Table-2017 (ICMR-NIN), the fruit's 100 g edible portion contains a good amount of water content (moisture 92 g), both soluble and insoluble fiber (total 2.07 g), and total free sugar 3.42 g. Further, it has a significant amount of folate (15.38 mcg), biotin (2.23mcg), and Phylloquionone (K1-26 mg). In the categories of vitamins and minerals, almost all the B-vitamins in proportionate quantities, 47mg ascorbic acid, 26mg calcium, and a very high amount of potassium, i.e., 202 mg in 100 g edible fruit. As per IFCT-2017, Citrus limetta also rich in good numbers of fatty acids: total PUFA 86 mg, total SFA 78 mg, 68 mg linoleic acid, and 66 mg palmitic acid.Similarly, aspartic acid 20 g, glutamic acid (5.5 g), proline(6.9 g), amino acids are found remarkable. Above all, total polyphenols 117 (+/-12 mg) is highest amongst all thecitrus fruits available in tropical countries like India.11, 12, 13
Table 1
Nutrients and bioactive components (BAC) of sweetlime (Citrus limetta)14
Table 2
A list of all RNA viruses with their structure and tentative role of BioActive Compounds present in sweet Lime.15, 16, 17, 18, 19, 20, 2122, 23, 24, 25, 26, 27, 28, 29
Table 0
Table 1 Refers to nutrients and bioactive components (BAC) of sweet lime (Citrus limetta).13
Indian diets and sweet lime
Indian sweet lime (Mosambi) is under the Rutaceaefamily is one of the best rich sources of vitamin C, naringin, hesperidin, flavanones, anthocyanins, and other polyphenols that altogether boost immunity, antioxidant, anti-inflammatory, and total health benefits effects. There are various diet patterns across the world, out of which vegetarian, non-vegetarian, and Mediterranean diets are becoming popular in addition to typical Ketogenic, Paleolithic, Atkin, Dukan diets. In India, large numbers of the population are eithervegetarian or vegan. People from the north and west, and middle parts of India, thoroughly practice vegetarian diet patterns, while Jain people are typically vegan. In the same way, people from the east, north-east, south usually choose a non-vegetarian or ovo-vegetarian diet.30, 31, 32 Whatever the reasons behind diet practice, seasonal Indian fruits effectively combatdifferent lifestyle disorders like obesity, diabetes, and skin or hair issues. Vegetables, fruits, and citrus fruits are rich in bioactive substances such as minerals, polyphenols, and vitamins, including vitamins A, C, D, E. Hence, sweet lime can be named one of the best Indian superfoods due to its all-out potentialities.33, 34, 35, 36
Pathophysiology of SARS-CoV-2
Structure of SARS-CoV-2
The main focus of this review study is the most concerned RNA virus, SARS-CoV-2, which belongs to Betacoronavirus (genus), Sarbecovirus (subgenus), and Coronaviridae (family). SARS-CoV-2 is the virus that causes COVID-19 (the disease), usually spread by droplets during coughing or sneezing of an infected person. The COVID-19 or Coronaviruses are enveloped, positive-stranded RNA viruses with spike-like glycoprotein projections on their surface that look like a crown. Several structural and non-structural proteins are encoded in the coronavirus genome. The structural proteins are membrane (M), the envelope (E), and the spike protein (S) in charge of host infection, membrane fusion, viral assembly, morphogenesis, and virus particle release. The non-structural proteins (nsps; 3, 5, 11, 14, and 15) make viral replication and transcription easier.37, 38, 39, 40
Life cycle of the virus
Attachment, penetration, biosynthesis, maturity, and release are the five processes in the virus's life cycle with its host. Viruses bind to the host receptor by attachment, followed by penetration through membrane fusion or endocytosis. Viral RNA reaches the nucleus for replication when the viral contents are released into the host cells. Similarly, viral proteins are made from viral mRNA, called biosynthesis. As a functional receptor, Angiotensin-converting enzyme 2 (ACE2) was found in various tissues, including lung cells, gastrointestinal tissue, and even the brain. The virus's spike glycoprotein mediates SARS-CoV-2 internalization to its receptor (ACE2) on cell membranes of the organs in the human body.41, 42, 43, 44, 45
COVID-19 Inflammatory markers
Gao et al.,2020 discussed in their paper that COVID-19 is dependent on inflammatory mediators. Its replication causes inflammatory responses in cells, macrophages, and the release of cytokines. Several inflammatory markers can be used to identify and detect disease specificity. Inflammatory markers like procalcitonin (PCT), serum ferritin, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), interleukin-6 (IL-6), and serum amyloid A (SAA) have been correlated to a higher risk of COVID19.45, 46, 47, 48, 49
Phopholipid and functional enzymes
Abdalla et al. 2020 reviewed in their article that the primary components of the pulmonary surfactant are phospholipids, with phosphatidylcholines (PCs) being the most common. COVID-19 infection caused an increase in cytosolic phospholipases A2 (cPLA2) expression, resulting in phosphatidylglycerol depletion and cleavage of esterified fatty acids. It also increases inflammation and lung damage. Secretory phospholipase A2 (sPLA2) is another enzyme involved in eicosanoid synthesis. Monocytes, macrophages, neutrophils, and eosinophilsincrease and produce inflammatory substances like cytokines and chemokines.47, 48, 49, 50, 51
RNA mechanism and infection process
The viral particle gets entered into a small structure outside or inside a cell of the human body. The vesicle's envelope is removed due to the viral particle and welcomes the genomic RNA that usually gets released into the cytoplasm. The two types of Open Reading Frame, also called ORF1a and ORF1b RNAs, are produced by the genomic RNA, followed by the translation into polyproteins of the virus (pp1a and ppa1b). After this stage, a proteolytic process operated by viral enzymes breaks down the pp1a and ppa1b proteins. The action results in a total of 16 non-structural proteins. In contrast, some non-structural proteins form a transcription or replication complex (RNA-dependent RNA polymerase) that uses genomic RNA positive (a typical model). Subgenomic RNAs produced through transcription are structural proteins after translation, will form new viral particles.43 When the viral load is high, cell is infected with many viral particles. As a result, the cell's entire protein synthesis machinery is devoted to viral replication before the cell dies. The final step may be triggered by the "apoptosis" process (if death is slow and controlled) or by "energetic-metabolic chaos," which results in the breakdown of cell membranes, including lysosomes, and a complete loss of structural integrity. T-lymphocytes and antibodies may strike the infected cell and autoimmune phenomena.52, 53, 54, 55
Role of bioactive compounds in sweet lime towards RNA viruses56, 57, 58, 59, 60, 61, 62
This short review draws attention to some constituents of sweet lime (Citrus limetta) for their multivitamin and flavonoid content. Among the flavonoids, hesperidin has the most crucial role.
Hesperidin
Hesperidin is a plant-sourced pigment, aflavanone glycoside found in almost all citrus fruits, and plays a tremendous antioxidant activity. Out of total polyphenols, its contribution is maximum (13.45 mg) in 100 g edible mosambifruit pulp or fruit juices. This compound so farhas attracted the attention of many researchers because of its binding capacityto the spike protein and protease of the RNA viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It alters the polyproteins of the virus (pp1a and ppa1b) into the complex genomic structure responsible forviral replication. A study showed that hesperidin and ascorbic acid inhibit or slow down the formation of free radicals and reduce infections and inflammation.Hence, it is one of the best components in the fruit.57, 58, 59, 60
In COVID-19 patients, there is a possible chance that hesperidin interacts better with the SARS-CoV-2 protease. However, other lime flavonoids with lower binding energy (compared to the reference ligands, lopinavir, and nafamostat) to the three essential proteins, such as tangerine, naringenin, and nobiletin, also have low binding.However, considering the mode of action of this BAC, mosambiwhole fruit or juice may be helpful towards RNA virus infections.55, 56, 57, 58, 59
Naringenin and naringin
Naringenin is the metabolite of naringin and anaglycone.Naringenin is present in pure juice, whereas naringin is in citrus juices. Naringin is a glycoside under the flavanone group. It contributes in mosambi 1.64 mg in 100 g edible fruit pulp or fruit juice out of total polyphenols. There is a difference between these two flavanones that the sugar moiety in naringin induces steric hindrance of the scavenging community, making it less potent than naringenin. The water solubility of naringin is mild, so in the intestine, the gut microflora converts naringin to its aglyconnaringenin, which is better absorbed. Naringin in sweet lime (Citrus limetta) is metabolized in the proximal colon by GUT microbiota as good bacterias with the production of their hesperetin, aglycones,other phenolic compounds. Studies have also shown that intestinal flavonoids and their metabolitesregulate the constituents and activity of the intestinal bacterias, which ultimately results in sound physiological effects in the GI tract. Overall the compounds have a highly decisive inhibitory role against RNA virus infections.61, 62, 63, 64
Hesperetin
Hesperetin is the flavanone group found abundantly in all citruses,including sweet lime. The fruit's100 g edible portion contains 22.17 mg of this flavanone, one of the best bioactive compounds responsible in mosambi fruit or juice for its potent antioxidant activities. Hesperetin and its metabolites play a critical role in reducing inflammatory conditions through various anti-inflammatory pathways in the human body. Glycosidichesperetin, also called hesperidin, provides significant responses tothe anticarcinogenic effect. Further, hesperetin administration resulted in higher expression of various detoxifying enzymes in rat models of chemically induced colon cancer and rat models of lung cancer. In rat hepatocytes, hesperetin prevented bile acid-induced apoptosis and cytokine-induced inflammation. TUNEL assay and serum AST and ALT levels revealed that hesperetin improved liver histology and protected against hepatocyte injury.65, 66
Chlorogenic acid
CGA is a group of phenolic secondary metabolites formed by some plant species and is an essential component of coffee. It is one of the most abundant polyphenol compounds in the human diet. Antibacterial, antioxidant, and anticarcinogenic effects are just some of the biological properties of CGA. Its functions and applications, especially about glucose and lipid metabolism, have recently received increased attention.CGA inhibits the activity of α-glucosidase, G-6-pase expression, and HMG COA reductase.Hence, its various essential functions may help COVID-19 and its comorbid patients. The most crucial parts are lipid profile alterations by balancing lipoproteins, enzymes involved in lipid metabolism, fat absorption inhibition, and activation of fat metabolism in the liver. Further, it plays a significant role in reducing the susceptibility of LDL oxidation and decreasing LDLcholesterol. Well-controlled secretion of insulin, improving glucose tolerance and resistance. Hence, it has one of the best practical hypoglycemic and antidiabetic effects through mosambi or other citrus fruits for any RNA virus-infected patients.65, 66, 67
Ferulic acid
Out of total polyphenols in sweet lime, although ferulic acid is found (0.02mg) inminimal quantities, it also plays remarkable inhibitory and anti-inflammatory roles against viral diseases. It acts inside the fruit as a potent antioxidant, antimicrobial, antiallergic, hepatoprotective, anticarcinogenic, antithrombotic, increases sperm viability, antiviral, and vasodilatory actions, metal chelation, regulation of enzyme activity, activation of transcriptional factors, gene expression, and signal transduction are just some of the biological activities. 65, 66, 67, 68, 69
Zinc
Zinc is an essential element for all people of different ages. Although it is deficient in sweet lime,it still helps in cognitive impairment, growth, neurosensory disorders, hyperammonemia, and immunity. A study showed that zinc enhances serum testosterone level, oligospermia by improving T-cells function.69, 70, 71, 72
Conclusions
Sweet lime (Citrus limetta) is one of the best citrus fruits having almost all vitamins, minerals, amino acids, fatty acids, and polyphenols. It has the 2nd highest total polyphenols, with a high amount of potassium among citrus groups. Hence, considering multiple nutrients and plenty of bioactive compounds, it is usually a potent antioxidant. In general, sweet lime pulp and juice can be consumed regularly for their inhibitory and complementary therapeutic effect against RNA viruses, including SARS-CoV-2, except for chronic kidney diseases or other critical health issues. More clinical trials are needed to prove its efficacy; however, reviewing current knowledge, it is one of the potent antioxidant, inflammatory super food available and affordable almost worldwide.
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