Catunaregam spinosa (Thunb.) Tirveng: A Review of Traditional Uses, Phytochemistry, Pharmacological Activities, and Toxicological Aspects

Catunaregam spinosa (Thunb.) Tirveng. (Syn. Randia dumetorum (Retz.) Lam.), belonging to the Rubiaceae family, is distributed in south Asian countries. It is used as a traditional medicine to treat gastrointestinal and hepatic problems and as an anti-inflammatory and antimicrobial agent. The main aim of this review is to collect and analyze the available scientific information on traditional uses, phytochemistry, and pharmacological activities of C. spinosa. The scientific information related to C. spinosa was collected from various resources and databases such as SciFinder, Scopus, PubMed, and other databases. C. spinosa was found to be an important crude drug of the traditional medicinal systems such as Ayurveda. It was found to be used by the people of India as an alternative medicine, while the fruit of this plant was found to be used in dietary regimens as well. Active phytochemicals such as catunarosides, randianin, and several other saponins and triterpenoids possess various pharmacological activities such as anti-inflammatory, hepatoprotective, antibacterial, and immunomodulatory activities. Many studies have been performed to isolate the active compounds; however, there is a need for more activity-guided isolation studies. Various in vitro studies showed promising results but there are not many studies related to mechanism of actions using animal models. Hence, future studies on C. spinosa should focus on correlating the traditional uses with active phytoconstituents and modern pharmacological activities.


Introduction
Humans have been dependent on plants since the ancient times for various reasons such as food, shelter, and medicine [1]. Medicinal plants are the primary source of health benefits in various communities of the world [2]. More than 80% of the world's population is reported to use plant-based medicines as primary healthcare as they are easily available and safe to use [3]. Recently, researchers have developed traditional knowledge about medicinal plants which can fulfill the current gap in therapeutics, nutraceuticals, drug discovery, and development [4]. Especially in developing countries, the reliance on plant medicine is a typical basis for addressing the ethnomedicinal knowledge in therapeutics from different ethnic communities [5].
Altogether 68 names of species are recorded under the genus Catunaregam belonging to family Rubiaceae [6]. e particular species Catunaregam spinosa ( unb.) Tirveng. (Figure 1) is a shrub with thorns which is distributed up to 4000 ft. from the sea level [7]. e other synonyms of this species are given in Table 1 [6]. C. spinosa, known as Madana in Sanskrit, Madanphal in Nepali, and emetic nut or mountain pomegranate in English, is a deciduous shrub with approx. 5 m of height, leaves are ovate, simple, shiny, and pubescent [8], and flowers are white solitary and possess a honey-like fragrance [9]. is plant has been reported from various parts of India [10], Nepal, Bangladesh, South China [11], and the African subcontinent [12]. C. spinosa is reported to be distributed in tidelands of semitropical and subtropical areas [13]. e ancient medicinal systems of Ayurveda and Siddha use this plant to treat various types of symptoms and it is still in use [14]. "Rasayana," a kind of clinical specialties in Ayurveda, uses this plant for promoting good habits in the dietary regimen [15]. People in India and Brazil use this plant for treating food poisoning, inducing vomiting, and treating allergy and inflammation [16]. e presence of various important phytoconstituents such as phenols, flavonoids [17], triterpene saponins [18,19], hemolytic saponins, randianin [20], iridoids [21], dihydroisocoumarin [13], and many more other phytoconstituents is reported from various parts. Several studies have reported the modern pharmaceutical activities of C. spinosa such as piscicidal [22], molluscicidal [23], antioxidant [8], anti-inflammatory, antidiabetic, and antihyperlipidemic [24] activities. e isolated compounds are not well studied for the possibility of drug discovery pathways. It is important to have a clear idea of this medicinally important plant and its scientific progress.
e review of the current state of C. spinosa and its overall perspective related to ethnomedicinal importance and modern medicine is still lacking.
us, this review aims to collect and analyze the current status of traditional uses, phytochemicals, and pharmacological activities from the available scientific information.

Methodology
e scientific information of C. spinosa was collected from various online databases such as SciFinder, Scopus, Google Scholar, PubMed, Web of Science, and ScienceDirect. Additionally, the books, proceedings, comments, and editorials were used as secondary sources. e scientific names and their synonyms were cross-checked from the database available in the website of " e Plant List" (http://www. theplantlist.org/). e articles of better quality with sufficient taxonomical, ethnomedicinal, and pharmacological information were selected while preparing this review.

Traditional Uses in Medicine and Food
e fruit of C. spinosa is traditionally used to treat various symptoms (Table 2). Almost all parts of the plant have been used as a traditional medicine in Ayurveda and fruits have been reported to be used in medicine as well as in food. is plant is reported to treat ulcers, inflammation, tumors, wounds, and skin diseases [7]. People from the upper hilly region of India used its bark for curing diarrhea and dysentery [25]. e fruits or pulps of this plant are used as an abortifacient [26] and nauseant [11], as well as for treating asthma [27,28]. e fruit of C. spinosa is also taken as a nutrient or diet.
ere have also been studies on nutritional contents of the fresh fruit of this plant and they were reported to contain a high amount of carbohydrate [38]. 11-Methylixoside was isolated from the bark of C. spinosa [46]. e bioactive compounds are enlisted in Table 3 and their structures are in Figures 2-6.

Pharmacological Activities
Due to the widespread traditional uses, C. spinosa extracts and compounds have been subjected to various pharmacological activity evaluations. is plant has been studied widely for its pharmacological activities, mainly antibacterial, antioxidant, anti-inflammatory, antimicrobial, anticancer, and antifertility activities. Few studies have reported sedative and analgesic activities. Most of the activities are based on random screening rather than traditional knowledge, which cannot provide the correlation between ethnomedicine and modern pharmacological activities. Some studies carried out on C. spinosa are discussed in the following section.

Antimicrobial Activities.
e extract obtained from the fruit bark of C. spinosa showed prominent antibacterial activity against various species of Proteus, Staphylococcus, Clostridium, Salmonella, Vibrio, Bacillus, Escherichia, and Pseudomonas [53]. e water extract of the seed of C. spinosa was tested against human pathogenic bacteria such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis; and the minimum inhibitory concentration (MIC) was found to be 0.07, 0.08, 0.88, 3, and 0.73 mg/mL, respectively, and the zone of inhibition ranged from 15 to 20 mm [36].
In another study, the antimicrobial activity was performed by the broth dilution method and disc diffusion method against various Gram-positive and Gram-negative bacteria.
e methanolic extract of leaves of C. spinosa showed a potent effect against Gram-negative bacteria, that is, K. pneumoniae, E. coli, and S. typhi, as compared to Gram-positive bacteria, that is, B. subtilis and S. aureus [40]. Evidence-Based Complementary and Alternative Medicine e fruit extract of C. spinosa was subjected to its antimicrobial activity by the disc diffusion method, where it was tested against P. aeruginosa, E. coli, S. pyogenes, S. typhi, S. pyogenes, and B. subtilis. e activity of the extract was tested as compared to the standard Chloramphenicol. One hundred µg/mL of the ethanolic extract showed more potency and highest zone of inhibition of 28.0 mm against the Bacillus and Klebsiella pneumoniae of 24.0 mm and the lowest was that of Streptococcus pyogenes. In chloroform extract, the maximum zone of inhibition of 26 mm was recorded for Bacillus subtilis followed by Pseudomonas aeruginosa of 16 mm and it was lower for the remaining bacteria. In the aqueous extract, the maximum zone of inhibition of 18 mm was recorded for Klebsiella pneumonia followed by Bacillus subtilis of 14.0 mm and it was lower for other bacteria [54].

Hepatoprotective Activities.
e hepatoprotective potential of methanolic extract of the fruit of C. spinosa at the dose of 200 mg/kg was studied in male albino Wistar rats with alcohol (28.5%) induced liver injury. e biochemical parameter and histopathology of the liver were accessed before and after treatment and were found to be significant to retrieve the parameters towards the normal stage. e treatment with standard silymarin and C. spinosa extract showed a decrease in the elevated levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), direct bilirubin (DB), and triglyceride (TG) as compared to rats exposed to alcohol only. e histopathological examination showed severe necrosis in cells treated with alcohol only, while reduced necrosis was found in the rats treated with C. spinosa extracts [55].
A similar study was performed in carbon tetrachloride (CCl 4 ) induced hepatic damaged rats [17]. e animal treated with C. spinosa leaf and bark extracts at 400 mg/kg for 15 days showed a significant decrease in various biochemical parameters such as ALP, AST, ALT, LDH, albumin, and total and direct bilirubin and an increase in total protein levels as compared to the CCl 4 only treated group.
However, in these studies, the mechanism by which C. spinosa is protecting liver injury is yet to be studied. Further, the studies on the isolation and identification of active compounds and elucidation of their mechanism of action are necessary.

Antioxidant Activities.
Methanolic extracts obtained from the fruit bark of C. spinosa were studied for their antioxidant activities. e DPPH radical scavenging assay, nitric-oxide scavenging assay, and superoxide inhibition assay were done for which the value was expressed in EC 50 . e EC 50 value for the DPPH assay for the extract was 37 µg/ mL, whereas the EC 50 value for standard (pyrogallol) was 1.7 µg/mL. e EC 50 value of the extract for nitric-oxide scavenging assay was 16.8 µg/mL as compared to the    [53].
In another study to evaluate the DPPH free radical scavenging activity for the methanolic extract of leaves, the extract showed the IC 50 values of 45.12 µg/mL as compared to ascorbic acid (34.91 µg/mL). Similarly, in the ferrous ion chelating activity study, the extract had the IC 50 value of 64.70 µg/mL as compared to ascorbic acid (30.96 µg/mL) [56]. e DPPH and ABTS + scavenging assay were performed to determine the antioxidant potential of leaves of C. spinosa [40] . e IC 50 value recorded was 0.154 ± 0.14 and 0.175 ± 0.12 mg/mL, respectively. Another study recorded the IC 50 value of stem and leaves of C. spinosa as 8.08 and 5.35 mg/mL, respectively, as compared to the IC 50 value of ascorbic acid (<2 mg/mL) [39].
All these antioxidant activity evaluation studies were based on the in vitro analysis. e in vivo antioxidant properties and their mechanism of action are yet to be studied.

Anti-Inflammatory Activities.
Anti-inflammatory activities of C. spinosa were studied in the carrageenan-induced inflammation test in the hind paw of rats. e inflammation was inhibited by 31.69, 35.11, and 41.62% at 1, 3, and 5 h, respectively, by 200 mg/kg of a dose of ethanolic extract of leaves of C. spinosa, which was comparable to the inhibition of standard drug indomethacin (10 mg/kg) [57].        Evidence-Based Complementary and Alternative Medicine 5.5. Antihyperglycemic Activities. Antihyperglycemic activities of methanolic extract of C. spinosa fruit extract were studied in the streptozotocin-induced male Wistar diabetic rats. e group of rats treated with standard drug gliclazide and C. spinosa fruit extract showed a decreased level of glucose as compared to the diabetic control group. e value of blood sugar decreased from 88.5 ± 6.86 to 74.16 ± 4.02 in gliclazide (50 mg/kg) treated group in between 60 and 120 min, while C. spinosa fruit extract (400 mg/kg) decreased the blood sugar level from 106.16 ± 7.8 to 87.5 ± 5.00 mg/dL [24]. e detailed biochemical mechanism by which it acts against hyperglycemia is yet to be studied. e study of isolation of bioactive compounds which can act as hypoglycemic activity from C. spinosa and their characterization can lead to the development of potential drugs in the future.

Wound Healing Activities.
e wound healing was performed in the HUVECs cell and it was treated with catunaregin obtained from the plant C. spinosa at concentrations of 10, 50, and 100 μM and found to be significant for preventing cell migration. A maximum decrease of 65.4% in cell migration was recorded for 100 μM. e next invasion assay was done to demonstrate the antiangiogenesis activity. In this assay, catunaregin significantly decreased the invasion of HUVECs induced by VEGF. e highest decrease of the invasion of HUVECs (61.3%) was recorded for the concentration of 100 μM [31].

Anticataleptic Activities.
e anticataleptic efficiency of the ethanolic extract of fruits of C. spinosa was determined in the catalepsy in mice induced by clonidine and haloperidol. In this study, the forepaw of mice was placed in the bar and the time required to remove the paw from the bar was recorded. e catalepsy recorded for clonidine was 229.2 ± 14.88 sec., which was significantly decreased to 135.2 ± 4.84 seconds when treated with 720 mg/kg of ethanolic extract of fruits of C. spinosa similar to that of standard chlorpheniramine maleate [58].

Other Activities.
e anthelmintic activity of the fruit extract of C. spinosa was studied in Pheretima posthuma. e extract of C. spinosa (200 mg/mL) was tested against worms for its ability to paralyze and kill the worms. e study showed that the ethanolic extract revels comparable results similar to albendazole (10 mg/mL), a standard drug as a positive control [59].
ere are several other ethnomedicinal practices of C. spinosa such as treating ulcers, asthma, abortifacient,

Toxicological Aspects
Acute toxicity study showed that the aqueous and alcoholic extracts of C. spinosa at a dose of 2000 mg/kg in a Swiss albino mice model were found to be nonlethal [14]. Another study regarding the toxicity of 11-methylioxoside obtained from the bark of C. spinosa was reported by Jangwan et al. [46]. Upon administrating the single dose of 5000 mg/kg extract of C. spinosa in Wistar albino rats, no mortality was observed. However, when administrating the different doses of 200 mg/kg, 2000 mg/kg, and 5000 mg/kg continuously up to 14 days, the mortality observed was 0%, 25%, and 50%, respectively. e result was supported by a change in biochemical parameters and histopathology reports [60].
Brine shrimp lethality assay was done to study the cytotoxic activities of C. spinosa leaf extract and lethal concentration (LC 50 ) was determined. e LC 50 values of water and ethanol extract were 0.94 and 0.23 µg/ml, respectively [12]. A similar study was done in the methanolic extract of the stem and the LC 50 value was found to be 1.32 µg/mL [29].

Conclusion and Future Prospects
C. spinosa is reported to be used in traditional medicine as well as in dietary regimens. Almost all parts of the plant have been used in traditional medicines such as leaves, fruits, stem, stem bark, fruit bark, and root bark. Fruits are also used in food. Various classes of compounds such as polyphenols, flavonoids, terpenoids, triterpenoids, and saponins are reported to be the main constituents found in this plant. e diverse pharmacological activities such as anti-inflammatory, antimicrobial, antidiabetic, antioxidant, and hepatoprotective activities have been investigated with promising results. Despite those promising results, many studies are based only on the in vitro models, and mechanisms of action are not well studied. Some of the research lacked the proper use of positive and negative control in the experiment setting. e in vivo studies were performed with very high doses of extracts and seem practically less efficient. Some ethnopharmacological reports show that this plant has been used to treat ulcer symptoms and acidity; however, pharmacological activities were not performed to evaluate these activities. Future research work must correlate the traditional knowledge with the isolation of active constituents and corresponding pharmacological significance. Along with this, the details of toxicological activities if studied can pave the way to develop new therapeutic agents.

Data Availability
Data sharing is not applicable. No new data were generated.

Conflicts of Interest
e authors declare no potential conflicts of interest.

Authors' Contributions
DT conceived the idea and prepared the first draft of the manuscript. DB and KRS contributed to the literature survey. HPD contributed to the preparation of the first draft and revised the manuscript. All authors read and approved the final version of the manuscript before submission.