In vitro antioxidant, anti-inflammatory, and anticancer activities of methanolic extract and its metabolites of whole plant Cardiospermum canescens Wall

Traditionally, the whole plant of Cardiospermum canescens has wide applications in the management of oxidative stress and inflammation in Africa and Asia. The present study investigated the antioxidant, anti-inflammatory, xanthine oxidase (XO) inhibitory, and anticancer activities of metabolites present in the crude methanolic extract of whole plant C. canescens (CCE). Chemical examination of CCE revealed the presence of six known compounds (1–6). From the results of in vitro studies, it can confirm that CCE exhibited notably inhibition of DPPH and superoxide free radicals, along with COX-1, COX-2, 5-LOX, and XO enzymes. Compounds 2 and 3 showed significant inhibition of DPPH and superoxide free radicals. Also, compound 2 exhibited good inhibition of COX-1 and COX-2 enzyme with IC50 of 87.0 and 88.0 μg/mL. Furthermore, CCE exhibited significant inhibition of 5-LOX and XO enzymes with IC50 of 42.5 and 56.0 μg/mL, respectively, while standard with IC50 of 42.5 and 56.0 μg/mL, respectively. Among the test series of cancer cell lines, compounds 2, 3, and CCE showed a significant percentage of cell growth lysis of DLD-1 with IC50 values of 52.5, 72.5, and 32.5 μg/mL, respectively. Besides, all the metabolites and CCE showed a very weak degree of specificity against NHME, indicates less toxicity to normal cells. To conclude, the results of the present study indicated that the methanolic extract from the whole plant of C. canescens displayed antioxidant activity by inhibiting DPPH and superoxide free radicals; anti-inflammatory effects by regulating enzymes COX-1, COX-2, 5-LOX, and XO; and anticancer activity by inhibiting the growth of MCF-7, DLD-1, HeLa, and A549. These activities can link to natural active compounds 2 and 3. This study supports the traditional uses of the root of C. canescens. These data findings suggest that C. canescens can be a promising natural source of biological medicines for oxidative stress, inflammation, gout, and cancer.


Background
Cardiospermum is the sole genus of the tropical family Sapindaceae, which records in the flora of Africa and Asia. Cardiospermum genus comprises about 30 species across the globe; most of them are reported to be existing in India. Among these, Cardiospermum canescens is a climber, commonly termed as "Love in a puff" and "Balloon plant." [1] In the folklore of India and some other Asian countries, the whole plant of Cardiospermum species has been used in the treatment of swelling and tumors. Especially, the tribes of Africa and Asia used the whole plant of Cardiospermum in the management of rheumatoid arthritis [1,2]. Also, in the rural areas of Tamil Nadu state, India, the whole plant is used as a leafy vegetable for daily consumption [2,3].
Biologically, the whole plant of C. canescens reported for arthritis, lumbago, neuropathy, fever, stiffness of limbs, piles, nervous disorders, neuropathy, diaphoretic, snake bites, laxative, diuretic, emmenagogue, and mucilaginous [4]. Earlier, a series of phytochemical constituents, namely flavonoids, triterpenoids, saponins, carbohydrates, proteins, alkaloids, and tannins was identified from C. canescens [4,5]. To date, antioxidant, antibacterial, hepatoprotective effects, and cytotoxicity activities were investigated on C. canescens [1,3,5,6]. Besides, no chemical investigation has attempted to evaluate the chemical constituents of C. canescens. So, the present research study mainly aimed to analyze the chemical composition of the crude methanolic extract of whole plant C. canescens (CCE) employing chromatography and to monitor antioxidant, xanthine oxidase (XO) inhibitory, anti-inflammatory, and anticancer activities of isolated metabolites and CCE.

Plant material
The whole plant of Cardiospermum canescens Wall (Family: Sapindaceae) was collected in Tirupathi Seshachalam hills, Tirupati, Andhra Pradesh, India, in February 2019. Dr. K. Madhava Chetty of Faculty of Sri Venkateswara University (SVU), Tirupati, Andhra Pradesh, India, has authenticated the sample and a voucher specimen with accession number PS-2019-225 which has been deposited at the Department of Botany, SVU, Tirupati, Andhra Pradesh, India.
Extraction and isolation of compounds from whole plant C. canescens The whole plant C. canescens (1.0 kg) was dried and powdered and extracted thrice with methanol (96%) at 25°C (3 times × 3 days/time). The obtained fractions were evaporated under vacuum to obtain a methanolic extract of C. canescens (CCE, 45 g, 4.5%w/w).

Antioxidant activity DPPH assay
The metabolites (1-6) and CCE were subjected to 1,1diphenyl-2-picrylhydrazyl (DPPH) assay [7] in triplicate. Known concentrations of the sample added 0.004% DPPH in methanol. After that, it was incubated at 37°C for half an hour and recorded for absorbance at 517 nm against the blank using UV-visible spectrophotometry (Spectra MAX plus 384, USA).

Superoxide radical scavenging assay
The metabolites (1-6) and CCE were subjected to scavenging assay of superoxide [8] in triplicate. Known concentrations of the sample added 1 mL of a standardized solution containing 50 μM NBT + 73 μM NADH + 15 μM of PMS in phosphate buffer (pH 7.4) and incubated for 30 min. After that, absorbance was recorded at 562 nm against the blank.
In vitro assays of anti-inflammatory activity Cyclooxygenase (COX-1 and COX-2) inhibitory assay By COX inhibitor screening assay kit (Cayman Chemical Company, MI), metabolites (1)(2)(3)(4)(5)(6), and CCE were estimated for inhibitory activities of COX [9]. Diclofenac and different concentrations of extracts were used as inhibitor sources. The prostaglandin 2α amount obtained from COX-1 and COX-2 employing substrate arachidonic acid was estimated by using the enzyme immunoassay (EIA) of the same kit. This inhibitory assay performed thrice and their IC 50 values attained by analysis of linear regression.

5-Lipoxygenase inhibitory assay
The 5-lipoxygenase (5-LOX) activity [10] on linoleic acid was partly disrupted by using a series of metabolites (1-6) and CCE concentrations. By the end of the reaction time, the absorbances of reaction mixtures were determined at 234 nm and the IC 50 was then calculated. This inhibitory assay was performed thrice with Quercetin as a standard.

Xanthine oxidase inhibitory activity assay
The XO inhibitory activity [11] determined according to a described method with slight modifications. Briefly, to 10 μl of 5 mM xanthine (substrate), 470 μl of sodium phosphate buffer of pH 8.0, 10 μl of the sample (metabolites (1-6) and CCE) was dissolved in DMSO, and 10 μl of enzyme XO was added and mixed in the well, then incubated for 5 min at 25°C. Later, absorbance was noted at 295 nm. Allopurinol and DMSO were used as standard and control. This inhibitory assay was performed thrice, and their IC 50 values were obtained by plotting concentration against respective percentage inhibition.

In vitro anticancer assay Sulforhodamine B colorimetric assay
The in vitro anticancer activity of metabolites (1-6) and CCE was determined by Sulforhodamine B (SRB) assay [12] using four cancer cell lines-MCF-7 (Breast), DLD-1 (Colon), HeLa (Cervical), and A549 (Lung)-and one normal human cell line-normal human mammary epithelial (NHME) (provided by National Centre for Cell Science, Pune). All cell lines were maintained and sampling was prepared according to the procedures of Tatipamula et al. [12]. To 190 μL, screened ideal cell suspension was added to known concentrations of the sample and incubated for 3 h at 37°C with 90% relative humidity and in the presence of 5% CO 2 . After that, 100 μL cold TCA was added and again incubate for 1 h at 4°C. Then, the entire 96-well plate is washed and airdried at 25°C. Then, add 100 μL SRB solution (0.057%); after 30 min, rinse with 1% CH 3 COOH and add 200 μL Tris base (10 mM, pH 10.5) solution; and shake for 5 min; absorbance was measured at 510 nm against a blank (contains the only medium).

Statistical analysis
All in vitro assay test results were note as mean ± SD. A one-way analysis of variance (ANOVA) was followed by a t test with p < 0.05 measured to be statistically significant.

Chemistry
For the first time, six known compounds (1-6) were identified from the methanolic extract of C. canescens (CCE) utilizing chromatographic methods and analyses of spectral data namely 1 H NMR, MS, IR, and 13 C NMR

Antioxidant activity
Initially, CCE was exposed to an initial test against DPPH [7] and superoxide [8] assays, and its IC 50 values were found to be 60.0 and 62.5 μg/mL, respectively, whereas standard (ascorbic acid) value was 27.8 and 32.1 μg/mL, respectively. Based on the preliminary antioxidant analysis of CCE, we subjected its metabolites (1-6) for antioxidant activity. Among all the tested compounds, only compounds 2 and 3 showed moderate inhibition of DPPH and superoxide free radicals. The concentration of 2 needed for 50% inhibition of DPPH and superoxide free radicals was found to be 169.0 and 180.0 μg/mL, respectively, while 3 with 285.0 and 230.0 μg/mL, respectively (Fig. 2).

Discussion
In the present study, six known secondary metabolites (1-6) were isolated from the methanolic extract of whole plant C. canescens (CCE). This identification provides new information on the phytochemical profile of C. canescens. Also, the biological profile of C. canescens justifies its natural aptitude to act against free radicals, inflammation, and cancer. From the DPPH and superoxide free radical assay, it observed that CCE inhibits them prominently. In the deep enzymatic analysis of anti-inflammation proved that the methanolic extract of whole plant C. canescens has potent inhibition of COX-1 and 2, 5-LOX, and XO enzymes. This observation helps to investigate the anticancer ability of C. canescens, and the outcomes of SRB assay showed that CCE has a significant degree of specificity against MCF-7, DLD-1, HeLa, and A549. In general, inflammatory activity was caused by an elevated level of prostaglandins and leukotrienes in the body. Cyclooxygenase (COX-1 and 2) and 5-lipoxygenase (5-LOX) enzymes are responsible for the production of prostaglandins and leukotrienes, respectively. Hence, inhibition of COX-1 and 2 by anti-inflammatory drugs such as NSAIDs decrease the production of the prostaglandins, ultimately resulting in the reduction of inflammation, as well as pain [13][14][15]. Nevertheless, inhibition of particularly prostaglandins can cause activation of 5-LOX pathway alternative, which results in the elevation of proinflammatory and gastro-toxic leukotrienes production. Therefore, drugs that inhibit both COX and 5-LOX (dual inhibitors) reduce leukotrienes and prostaglandin production and completely inhibit inflammation. Also, by using dual inhibitors, the adverse effects on the cardiovascular system caused by selective COX-2 inhibitors (coxibs) will be reduced [16][17][18][19][20]. The results of the present study showed that CCE acted as a dual inhibitor and effectively inhibited both COX and 5-LOX enzymes, whereas compound 2 prominently inhibit particularly COX-1 enzyme.
Generally, uric acid formed from purines xanthine and purines hypoxanthine in the presence of an enzyme called xanthine oxidase (XO). Excess deposition of uric acid in the joints of the human body leads to severe joint pains, which is well known as gout (painful inflammation) [21,22]. Furthermore, XO is a good biological source for oxygen-containing free radicals that damage living tissues and cause-related inflammatory diseases like aging, cancer, and atherosclerosis. Hence, XO inhibitors play a vital role in diagnosis, not only gout but also related inflammatory diseases [23][24][25]. This study suggests that only CCE possesses XO inhibitory activity that might be helpful in the management of gout and other inflammatory-related diseases.
Many scientists reported that chronic inflammation is the key causative factor in various types of cancers. Usually, the longer the existence of the inflammation, the greater the risk of cancer. In general, chronic exposure to inflammatory mediators, which include free radicals, cytokines, COX-1, COX-2, and 5-LOX, leads to a sharp rise in mutagenesis, cell proliferation, and oncogene activation eventually leading to the proliferation of cells which lost control over normal growth [26][27][28][29][30][31]. So, as metabolites and CCE showed good anti-inflammatory properties, we further investigated their anticancer abilities using the SRB assay. From the outcomes, it is justified that compound 2, 3, and CCE have an ability to lysis the cells of MCF-7, DLD-1, HeLa, and A549. Also, all the metabolites and CCE are less toxic towards the human cell lines.
In the folklore, the whole plant of C. canescens has applications in managing acute arthritis, rheumatism, inflammation, and cancer in India. The present study scientifically justifies the uses of the C. canescens as an antioxidant, anti-inflammatory, and anticancer agent in traditional medicine. The major phytoconstituents in CCE were identified as 1-6. Thus, these active constituents might be responsible for the biological activities of CCE.

Conclusion
To conclude, the results of the present study indicated that the methanolic extract from the whole plant of C. canescens displayed antioxidant activity by inhibiting DPPH and superoxide free radicals; anti-inflammatory effects by regulating enzymes COX-1, COX-2, 5-LOX, and XO; and anticancer activity by inhibiting the growth of MCF-7, DLD-1, HeLa and A549. The key metabolite responsible for in vitro activities were claimed to be compound 2 and 3. The results provide evidence that supports the traditional uses of the whole plant of C. canescens. Also, these findings suggest that the plant of C. canescens can take an account as a good natural source of remedial medicine for alleviating pain and cancer. However, an in-deep chemical and biological examination is further required to use the natural source (i.e., C. canescens) in the management of oxidative stress conditions, inflammation, gout, and cancer.