Biological and phytochemical review on the genus Coccoloba (Polygonaceae)

Polygonaceae is one of the largest medicinal plant families, vastly distributed worldwide, containing around 1,200 species from 48 genera. Most of the species are located in the northern temperate region, while the other species are allocated from the tropics to the arctic. The prime genera in Polygonaceae are Eriogonum which includes 240 species, Rumex with 200 species, Coccoloba with 120 species, Persicaria with 100 species, and Calligonum with 80 species. Coccoloba is one of the most interesting genera of the family Polygonaceae in terms of biological activities and secondary metabolites. Plants of this genus are used worldwide in traditional folk medicine. The review is a comprehensive literature survey on different Coccoloba species regarding their biological activities and their isolated phytochemicals. Different classes of secondary metabolites were isolated from this genus including flavonoids, phenolic acids, tannins, triterpenes, diterpenes, anthraquinones, isochromene, and volatile oils. Crude extracts and isolated compounds of various Coccoloba species displayed diversity in biological activities. Further investigations are required to explore new bioactive compounds and their pharmacological activities.


INTRODUCTION
Plants are grouped in families based on their morphological, reproductive and genetic traits. Certain plant families possess interesting pharmacological activities targeting many humans' and animals' disorders. Polygonaceae is an interesting plant family with many genera that are utilized in conventional medicine all over the world across civilizations. Members of this family are distributed in almost every part of the world. It comprises 1,200 species from 48 genera, which can be found from the tropics to the arctic, while in the northern temperate region most of the species are flocked [1]. Among the most interesting genera of this family is Eriogonum which includes 240 species, Rumex with 200 species, Coccoloba with 120 species, Persicaria with 100 species, and Calligonum with 80 species [2]. The genus Coccoloba belongs to the tropical and subtropical areas of America, South America, the Caribbean, and Central America, with two species that extend to Florida. It comprises approximately 120-150 shrubs and trees, mostly perennials flowering plants, of which more than 25 plants occur in Cuba [3]. Coccoloba comes from the Spanish word "Coccolobis", a kind of grape or the Greek words "kokkos" means "berry, grain, or seeds" and lobos "pod or lobe" referring to the grapelike fruits [4].
The Rama of Southeastern Nicaragua used C. uvifera L. leaves and barks as an antidiarrheal remedy [5]. Bahamian island people used C. diversifolia berries for the treatment of diarrhea and reinforcement of physical endurance. It could be eaten and the bark extract is taken as an analgesic and anesthetic [6]. In Oaxaca, Veracruz, and Puebla, Mexico rural areas C. barbadensis leaves extracts are used for kidney problems [7]. Different Coccoloba species are used in Brazil as astringent, for the treatment of fever and diarrhea, menstrual disturbance, uterine hemorrhages, hemorrhoids and gonorrhea [8].
Native Americans used C. uvifera leaves, bark, and roots to make medicinal teas. Astringent decoctions and juices of the bark, wood, and roots of the plant were used to treat diarrhea, hemorrhages, dysentery, and venereal diseases. Externally, they are being applied for rashes and skin afflictions. Leaves were used for the treatment of hoarseness and asthma, and to wash wounds. Bark resinous gum was used against throat ailments, while the root decoction was used against dysentery [8]. C. mollis has been reported in folk medicine as beneficial in many cases as insomnia, memory loss, stress, anemia, diminishing eyesight and sexual impotency [9].
This review aims to summarize the reported biological and phytochemical studies of the genus Coccoloba. The data presented in this review were collected up to 2019 from various databases including SciFinder (https://scifinder.cas.org/SciFinder/login), Egyptian Knowledge Bank (https://www.ekb.eg/) and PubMed (http://www.ncbi.nlm.nih. gov/PubMed).

Genotoxic and mutagenic potentials
The ethanolic leaves and roots extracts of C. mollis were subjected to Salmonella/microsome assay (TA98 and TA100 strains, with and without exogenous metabolism-S9), as well as the comet and micronucleus tests. The results showed no significant rise in the number of revertants/plate of Salmonella strains in different concentrations analyzed of the root-extract, however, the extract was highly toxic itself to the Salmonella TA98 strain in the tests carried out with S9 (doses varying from 0.005 to 0.5 µg/plate). While, at the highest concentration assessed of the leaves extract the results showed induced mutations in the TA98 strain with the absence of S9, although it exhibited a very low mutagenic potency, 0.004 rev/µg. Furthermore, comets and micronuclei showed no statistically significant increase in their number, on using Swiss mice. So C. mollis extracts were not mutagenic, under the designed experimental conditions [9].

Antimicrobial activity
The antibacterial, antifungal, toxic and phototoxic activities were assessed for C. uvifera seeds methanol extract. The phytochemical content of the seeds extract was also investigated. The results showed the antibacterial effect of the extract against Staphylococcus aureus and Salmonella typhimurium. The ethyl acetate partition of the methanol extract (a brown precipitate) exhibited antibacterial activity against Gram-negative bacteria, Pseudomonas aeruginosa, and Escherichia coli, in addition to antifungal activity against Fusarium oxysporum, Candida albicans and Fusarium decencellulare [10].
An in vitro assay was done on the ethanol extract of C. acrostichoides aerial parts and different fractions for determining their antimicrobial activity. The extract displayed activity against the Staphylococcus aureus and Micrococcus luteus. Most of the fractions especially the n-hexane and ethyl acetate fractions also had an antifungal activity. Isolated β-sitosterol and betulin were tested for their antimicrobial activity. Betulin showed activity against Fusarium oxysporum [11].
Another comprehensive study investigated the in-vitro antimicrobial activity of Brazilian plant extracts through the disc diffusion method. Among the tested plants C. acrostichoides and C. cerifera showed interesting results. The aerial parts extract of C. acrostichoides showed 10.37-0.52 mm inhibition zone for Micrococcus luteus, and 7.17 ± 0.41 mm inhibition zone for Staphylococcus aureus, while the leaves extract of C. cerifera displayed 8.33±0.41 and 7.33±0.52 mm inhibition zone for M. luteus and S. aureus, respectively [12].
A study was done on the ethanolic bark extract of C. dugandiana to determine its antifungal activity. The extract exhibited an inhibitory effect on the growth of Cryptococcus neoformans. (-)-Epigallocatechin gallate and gallic acid were isolated from the extract through bioassay-guided fractionation. The biological testing results showed that (-)-epigallocatechin gallate inhibited C. neoformans with IC 50 = 1.6 µg/mL, and MIC= 12.5 µg/mL but showed no fungicidal activity. However, gallic acid was inactive [13].
The crude leaves extract of C. parimensis revealed an anti-plasmodium activity with IC 50 = 6−12 µg/mL through a novel DNA-based microfluorimetric method. The ethyl acetate fraction showed IC 50 =10 µg/mL. A methyl ester derivative of gallic acid was isolated on the purification of this fraction showing IC 50 values < 2 µg/mL [14].
The antifungal activity was tested for the C. mollis ethanolic extracts of the leaves and roots as well as the anthraquinones isolated from the roots of this plant against Botryosphaeria rhodina, Botryosphaeria ribis, Lasiodiplodia theobromae, and Fusarium species. The ethanolic extract showed promising fungicidal activity, whereas the most active compound was emodin, which displayed inhibition for the microorganisms tested up to 44% [15].
An antibacterial activity study was performed for plants used in Jamaican folk medicine through disk diffusion method and showed that C. krugii demonstrated weak activity against the Gram-negative bacteria, Proteus mirabilis, with inhibition zone 10-12 mm and moderate activity against the Gram-positive bacteria, Staphylococcus aureus with inhibition zone 12-14 mm [16].
Furthermore, the antitrypanosomal activity was evaluated for several plant extracts through measuring the inhibitory effect on the growth of trypomastigote blood forms of Trypanosoma brucei in a primary screening assay at concentration 20 μg/mL. C. pubescens stem extract was identified as one of the highly potent antitrypanosomal extracts with an IC 50 value of 0.83±83 μg/mL [17].

Anti-inflammatory activity
Nineteen plant extracts were assayed against TNF-α and CCL 2 release by lipopolysaccharide-(LPS-) stimulated THP-1 cells, a human monocytic leukemia cell line, along with their radical scavenging activity on, DPPH. C. cereifera (aerial parts), inhibited the production of TNF-α in a concentration-dependent order.

Cytotoxicity
The toxicological analysis was carried out using the brine shrimp lethal assay (BSLA). BSLA was measured as the median lethal concentration (LC 50 ) that kills 50% of the larvae within 24 hours of contact with the aqueous plant extract of C. uvifera showing LC 50 of 10071 μg/mL [19].
In vitro assay was done on ten plants traditionally used in Maya medicine for their anti-neoplastic activity through the LNCaP prostate cancer androgen-sensitive cell line as a biological model for PCa. The extracts were evaluated in phenotypic screening, with a concentration of 25 µg/mL as a fixed-dose. MTT assay was conducted on C. uvifera leaves methanolic and dichloromethane extracts which showed interesting cytotoxic and antiproliferative activity [20].
Marcela S. Tsuboy and co-workers conducted cytotoxicity, genotoxicity, and apoptotic assays on the ethanolic extracts of C. mollis leaves and roots. They used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay (MTT), micronucleus test with cytokinesis block, comet assay, and an in-situ test for apoptotic cells detection. The results showed that C. mollis roots extract had higher cytotoxic activity than the resulting extract from the leaves and that the alleviation observed in cell viability in the MTT assay was, at least in part, a result of apoptosis induction. In the comet assay both extracts at a concentration of 20 µg/mL induced DNA damage, but with no genotoxicity detected in the micronucleus test with any of the treatments carried out [21].
MTT colorimetric assay was done on C. peltata ethanolic extract of the leaves and its fractions. The chloroform fraction was the most potent cytotoxic fraction followed by the ethyl acetate fraction which displayed significant cytotoxic activity, while the ethanolic, n-hexane fractions and the remaining aqueous fraction showed moderate activity against all the tested cell lines [22].
Nelson performed an antimitotic activity for the isolated diterpene ent-kaur-16-en-15-oxo-18oic acid from the methanolic extract of C. acuminate seeds by G2 checkpoint inhibition bioassay using the human breast cancer cells. The diterpene compound showed an IC 50 of 9 μg/mL [23].

Photoprotective activity
An in vitro model was conducted to assess the effects of C. uvifera extract (CUE) on tumor necrosis factor α (TNF-α), interleukin-1α (IL-1α), and α-MSH production in human epidermal melanocytes under both basal and UV-stimulated conditions. The anti-tyrosinase and antioxidant activities were evaluated as well. C. uvifera extract exhibited anti-tyrosinase and antioxidant activities and showed an inhibition in the production of TNF-α, IL-1α, and α-MSH in UVstimulated melanocytes (P<0.01). Furthermore, CUE inhibited tyrosine kinase activity in cell culture under both basal and UV-stimulated conditions (P<0.001) [25].

Anti-hyperglycemic activity
The anti-hyperglycemic effect of the hydroalcoholic leaves extract of C. uvifera was determined on blood glucose levels through oral glucose tolerance test, in fasting normal and glucose loaded hyperglycemic rats. The antioxidant activity was performed using AAPH (2,2'-azobis 2 amidino propane dihydrochloride) test and nitric oxide radical scavenging activity. The extract induced a significant reduction, in the treated group, for the hyperglycemic group compared with the control group. It also inhibited hemolysis of erythrocytes induced by AAPH in a dose-dependent manner and exhibited an antioxidant power comparable to that of the butylated hydroxytoluene (reference drug). The extract also inhibited nitric oxide production and showed potent reducing power [26].
The anti-diabetic activity of the leaves ethanolic extract of C. peltata was investigated in three different parameters including hypoglycemia, glucose tolerance test and STZinduced diabetes mellitus in rats. The blood glucose levels in hypoglycemic activity, glucose tolerance test, and anti-hyperglycemia, which were raised in streptozocin (STZ) induced diabetic rats were minimized by the ethanolic extracts (400 and 800 mg/kg b.wt.) [22].

Antioxidant activity
The antioxidant activity of C. uvifera fruits was evaluated using several in-vitro antioxidant assays. The TEAC value of 2,2′-azino-bis(3ethylbenzthiazoline-6-sulfonic acid) ABTS radical assay was found to be 897.6 μM of trolox/100 g of sample, while DPPH scavenging activity was 22.8% of DPPH free radical scavenging, for the ion chelation activity the results were 11.3% of Cu 2+ , 23.9% of Fe 2+ , and finally a Fe 2+ -reducing power of 0.76 mg/mL [27].
El-Kawe evaluated the antioxidant activity of the ethanolic extract of C. peltata leaves and its fractions. The most active fraction was the chloroform fraction followed by the ethyl acetate and aqueous fractions as ABTS scavenger [22].
The antioxidant activity of C. cowellii ethanol leaves extract was evaluated. The results showed that it possessed antioxidant activity through hydrogen donating abilities, using DPPH scavenging activity which was 34.01% at a concentration of 50 µg/mL [3].

Phytochemical constituents
Genus Coccoloba is rich in phytochemicals including flavonoids, tannins, terpenoids, volatile oils. Their structures are illustrated in the  following tables (1-8).

Flavonoids
Flavonoids are one of the most significantly important plant metabolites owing to their various biological activities. They are reported to possess antioxidant and antimicrobial properties in addition to antimutagenic and anticarcinogenic activities [29]. Quercetin glycosides are found commonly in the family Polygonaceae. For the genus, Coccoloba four flavonoids were isolated from the leaves extract of C. uvifera, myricetin 3-O-rhamnoside which was also previously isolated from the leaves extracts of C. peltata and C. dugandiana, myricetin 3-O-glucoside, quercetin 3-O-rhamnoside, and quercetin 3-O-arabinoside (Table 1).

Sterols
Plant sterols are one of the essential components of the membranes of all eukaryotic organisms. They are either synthesized de novo or taken up from the environment. Their function is to control membrane fluidity and permeability, however, some plant sterols have a definite function in the transduction of the signal. Furthermore, sterols possess a structure similar to cholesterol and have the ability to lower plasma cholesterol and LDL cholesterol [31]. Three compounds β-sitosterol, β-Sitosterol-3-O-β-Dglucoside, and sitostenone were isolated from the genus Coccoloba (Table 2).

Tannins
Epigallocatechin gallate (EGCg) is a chief catechin component in green tea [39] and it is well known for its diversity of biological activities such as antioxidant activity [40]

Diterpenes
Diterpenes are a structurally diverse class of C20 natural compounds, vastly distributed in nature, and possess various biological and pharmacological activities [44]. Three diterpenes were isolated from the genus Coccoloba, entkaur-16-en-15-oxo-18-oic acid, trans-phytol, and royleanone. The C. acuminata crude extract gave a strong positive response in the G2 inhibition bioassay and the active compound was determined to be ent-kaur-16-en-15-oxo-18-oic acid (Table 5).

Phenolic acids
Phenolic compounds are prevalent in plants. They are of significant interest due to their antioxidant properties [45]. Three phenolic acids were isolated from different Coccoloba species including gallic acid, the methyl ester of gallic acid, and vanillic acid (Table 6).

Conclusion
A literature survey on the genus Coccoloba revealed different chemical constituents discovered from this genus. Flavonoids, triterpenes, diterpenes, phenolic acids, sterols, and volatile oils constitute the major classes of a phytochemical constituent of this genus. However, the more extensive phytochemical and biological investigation is needed to be carried out, as the genus and the Polygonaceae family are rich sources of bioactive constituents that contribute to a broad range of medicinal activities. This current review demonstrated various biological studies performed on different extracts and isolated chemical constituents from different species of Coccoloba. The review focused on the assessment of the antioxidant, antimicrobial, cytotoxicity, genotoxic and mutagenic properties, anti-inflammatory, hypoglycemic, photoprotective activities of Coccoloba sp. Many biological and phytochemical investigations were reported from genus Coccoloba, revealed in this review including, C. mollis, C. uvifera, C. acrostichoides, C. kurgii, C. dugandiana, C. parimensis, C. peltata, C. excoriata, C. pubescens, C. cereifera, and C. acuminate being the most phytochemical and biological studied species leaving a great field for further exploration of other species that have not been yet fully discovered. The present review provides a comprehensive understanding of the chemistry and biology of different Coccoloba sp., which may help in the innovation and discovery of new alternative medications for the treatment of various health problems and diseases.

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Availability of data and materials
All data generated or analyzed during this study are included in this published article in the main manuscript.