Phytochemical Analysis, In-vitro Antiproliferative, Anti-oxidant Part 2

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a-Glucosidase inhibitory assay

A dilution series of fractions was made, to yield concentrations of 100,200,300,400, and 500mg/ml. The reaction mixtures contained 0.1 ml 1U/ml α-glucosidase solution mixed with 0.2ml of an extract dilution and 0.5 ml 100mM phosphate buffer, pH6.8. The mixtures were incubated at 37℃ for 15min. Then, 0.2ml 5mM p-nitrophenyl α-D-galactopyranoside PNPG was added to the reaction mixture, and incubation was extended for a further 20 min at 37℃. The reaction was terminated by adding 0.1 M NazCO3. The absorbance was recorded at a wavelength of 405 nm for all samples. Acarbose was used as a positive control at the same concentrations as the plant extracts. The results were expressed as percentage inhibition according to the following equation:

where Ag is the absorbance without an enzyme inhibitor, and As is the absorbance in the presence of the enzyme inhibitor [26].

Cell lines and MTS assay

HeLa and MCF7 cancer cell lines were obtained from ATCC, the cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum,1% penicillin/streptomycin antibiotics, and 1% L-glutamine. Cells were grown in a humidified atmosphere with 5% CO2 at 37°C. Cells were seeded at 2.6×10* cells/well in a 96-well plate. After 48h, cells were confluent, the media was changed. Cells were then incubated with different leaf fraction concentrations ranging from 0.25-10 mg/ml of R. rothschildianus for 24h. Cell viability was assessed

with the CellTilter 96°Aqueous One Solution Cell Proliferation (MTS) Assay according to the manufacturer's instructions (Promega Corporation, Madison, WI). Briefly, at the end of the treatment, 20 ul MTS solution per 100μl media was added to each well and incubated at 37°C for 2h. Absorbances were measured at 490 nm [27,28].

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Statistical analysis

All of the obtained results of the four studied plant fractions (antioxidant, anti-lipase, anti-amylase, anti-glycosidase, and cytotoxicity activities) were expressed as mean±SD standard deviation; the result was considered significant when the p-value was <0.05. Data were compared using unpaired t-tests.

Results

Phytochemical screening

The results of the preliminary phytochemical tests on the R. rothschildianus aqueous fractions showed the presence of saponin, phenols, protein, starch, and flavonoids. The methanol extract showed the presence of complex polysaccharides, phenols, protein, and flavonoids, while phenols, tannins, and flavonoids were observed in the acetone fraction, and in hexane fraction phenols, tannins, and flavonoids, glycosides, terpenoids, and steroids were identified as shown in Table 1. However, for the extraction process, methanol showed the highest percentage yield at 29.4%, followed by the acetone fraction at 16.5%. The aqueous extraction yielded 10.6%, while the lowest yield (7.3%) was in the hexane fraction(Table 2).

Quantitative analysis of TPC, TFC, and TTC For the evaluation of TPC, TFC, and TTC, the absorption (Abs) values of several concentrations of the gallic acid, rutin acid, and catechin standards (STDs) were obtained, and regarding these points, three equations were obtained for each STD versus its concentrations to calculate the total phenol, flavonoid, and tannin contents of the hexane, acetone, methanol, and aqueous R. Rothschild-diagnose fractions are presented in Table 3.

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Antioxidant activity

The results of assessing the free radical scavenging activity of four fractions from R. rothschildianus leaves, using Trolox as a potent antioxidant standard reference, were expressed as percentage DPPH inhibition (Fig. 1 and Table 4). Therefore, R. rothschildianus leaves could be considered an herbal source for antioxidants, and for the acetone fraction, which showed an ICso value of 6.3±0.4 μg/ml. Similar results were also obtained for the hex-ane fraction， which had an ICso value of 7.9±1.3μg/ml (Table 4). The results were compared to Trolox, a potent antioxidant compound, with an also equal to 3.1±0.9 μg/ml. By contrast， the aqueous fraction only showed moderate antioxidant activity, with a higher ICso value of 19.9±0.7μg/ml， while the methanol extract was in-active in this assay.

Lipase inhibition activity

In this assay, the anti-obesity activity of fractions from R. rothschildianus leaves extract was compared to that of

orlistat, a potent lipase inhibitory agent (Fig. 2 and Table 4).R. rothschildianus leaves were an excellent alternative natural source of lipase inhibitory agents. The acetone fraction showed an ICso value of 26.3±0.6μg/ml, which was very close to that of the reference com-pound orlistat （12.3±0.3μg/ml）. Hexane and methanol fractions only recorded moderate ICso values, equal to 39.8±0.3μg/ml and 60.3±0.4μg/ml， respectively； while the aqueous fraction was inactive.

a-Amylase inhibition activity

In this assay, the anti-amylase activity of fractions from R. rothschildianus leaves extract was compared to that of acarbose, a potent a-amylase inhibitory agent (Fig. 3). The acetone fraction was the most potent inhibitor of α-amylase， with an ICso of 19.0±0.7μg/ml， compared to 28.8±1.2 μg/ml for acarbose， the reference compound. This suggested that R. rothschildianus might be a powerful herbal remedy for diabetes. The aqueous fraction only showed moderate activity in this assay with an ICso value of 45.7±0.3 μg/ml， while hexane had an ICso value of 354.8±1.2μg/ml. The methanol fraction was inactive against the α-amylase enzyme. also, values were calculated for the four fractions(Table 4).

a-Glucosidase inhibition activity

Results for a-glucosidase were compared with those for acarbose, a strong enzyme inhibitory agent, and also values were calculated for the four fractions (Table 4 and Fig. 4). The acetone fraction exerted the greatest inhibitory action on α-glucosidase with an ICso of 54.9±0.3 μg/ml， compared with that of acarbose， the reference compound， at 37.1±0.3 μg/ml. By contrast， the methanol extract fraction showed only moderate inhibition of α-glucosidase， with an ICso of 251.2±0.4μg/ml， while the hexane and aqueous fractions were inactive in this assay.

Anti-proliferative activity

The results of treatment of HeLa and MCF7 cancer cells with five different concentrations in mg/ml for different extracts showed that the activity against the HeLa cancer cell line was better than against the MCF7 cancer cell line. However, hexane extract was the most potent

extract at 4mg/ml concentration with inhibition percentages of 98.9 and 97.4% against HeLa and MCF7 cancer cell lines, respectively, while methanol extract at the same concentration showed potent activity against HeLa and MCF7 cancer cell lines with 97.2 and 95.6% inhibition percentage. HeLa cell percentage inhibition on exposure to fractions from R. rothschildianus leaves was documented, compared to the positive control doxorubicin (Fig. 5a). MCF7 cells percentage inhibition was similarly determined for the four fractions from R. rothschildianus leaves, compared to the control (Fig. 5b).

Discussion

The DPPH radical scavenging assay is well known as a simple method for detecting antioxidant capacity in com-pounds. DPPH is a stable free radical that gives a purple color in alcohol solutions, and on reduction in the presence of hydrogen donating antioxidants, turns the solution colorless [29]. Therefore, R. rothschildianus leaves could be considered a natural source of antioxidants, especially for the acetone fraction, which showed an ICso value of 6.3±0.4μg/ml. Similar results were also obtained for the hexane fraction， which had an ICso value of 7.9±1.3 μg/ml (Table 4). The results were compared to Trolox, a potent antioxidant compound, with an also equal to 3.1±0.9 μg/ml. By contrast， the aqueous fraction only showed moderate antioxidant activity, with a higher ICso value of 19.9±0.7 μg/ml， while the methanol extract was inactive in this assay. These results were consistent with the presence of phenols and flavonoids in the plant, a powerful scavenger source for free radicals as shown with DPPH in this study. These results were in line with the diversity of phenolic compounds in plants, simple phenols like gallic acid, and more sophisticated phenolic acids like anthocyanins, hydroxyl cinnamic acid derivatives, and flavonoids. All these classes of compounds have received extensive attention due to their multiple physiological functions, especially free radical scavenging, anti-mutagenic, anti-inflammatory, and anti-carcinogenic activities [30]. As listed in Table 3, the acetone extract recorded the highest content of both phenolic compounds and flavonoids, 28.2±0.8mg of GAE/g and 107.3±4.6mg of RU/g, respectively. A previous report on some Rumex species found phenolic compounds in an ethanol extract from leaves of Rumex vicarious L., which were possibly involved in free radical reactions, reducing the stable used DPPH radical to a yellowish colored diphenyl picrylhydra-zine derivative from its original violet color [31].

R. rothschildianus leaves were an excellent alternative natural source of lipase inhibitory agents. The acetone fraction showed an ICso value of 26.3±0.6μg/ml， which was very close to that of the reference compound orlistat （12.3±0.3μg/ml）. Hexane and methanol fractions only re-corded moderate ICso values， equal to 39.8±0.3 μg/ml and 60.3±0.4μg/ml， respectively； while the aqueous fraction was inactive. Pancreatic lipase is a major enzyme involved in enterocyte triglyceride absorption. Therefore, its inhibition represents an important strategy in the management of obesity [32]. Plants rich in phenolic compounds have been screened in several reports for anti-lipase activity. Lipase inhibitory activity ranging from 40 to >70% has been found by in vitro tests in many different families, including Solanaceae (Solanum tuberosum), Brassicaceae (Brassica nigra and Raphanus sativus), Rosaceae (Malus Domestica Borkh. and Filipendula ulmaria (L.) Maxim.),

(Arctostaphylos uva-ursi (L.) Spreng. and Ericaceae Vaccinium myrtillus L.), and Fabaceae (Pisum sativum L. and Phaseolus vulgaris L.)[33].

The acetone fraction was the most potent inhibitor of α-amylase， with an ICso of 19.0±0.7μg/ml， compared to 28.8±1.2 μg/ml for acarbose， the reference compound. This suggested that R. rothschildianus might be a powerful herbal remedy for diabetes. The aqueous fraction only showed moderate activity in this assay with an ICso value of 45.7±0.3 μg/ml， while hexane had an ICso value of 354.8±1.2 μg/ml. The methanol fraction was inactive against α-amylase. A possible explanation for the aqueous fraction being a good enzyme inhibitor was the presence of saponins. Earlier scientific investigations found that saponins were bioactive against diabetes [34]. The potent effect of the acetone extract fraction against amylase might be due to the high content of both phenolic compounds and flavonoids. Corchorus olitorius experts α-amylase and α-glucosidase inhibitory effects due to constituents, especially phenolic compounds like caf-feic acid [35].

The acetone fraction exerted the greatest inhibitory action on a-glucosidase with an ICso of 54.9±0.3μg/ml， compared with that of acarbose, the reference compound， at 37.1±0.3μg/ml. By contrast， the methanol ex-tract fraction showed only moderate inhibition of a-glucosidase， with an ICso of 251.2±0.4μg/ml， while the hexane and aqueous fractions were inactive in this assay. The third category of oral hypoglycemic agents includes α-glucosidase inhibitors. There are a variety of α-glucosidase inhibitors, such as acarbose and voglibose, which usually are found in plant sources. They show valuable stabilization of blood glucose levels after a meal and have been used clinically in the management of diabetes mellitus [36, 37].

The results of treatment of both cancer cells (HeLa and MCF7) with various concentrations in mg/ml for different extracts showed that the general activity against the HeLa cancer cell line was better than the MCF7 cancer cell line. The hexane extract showed potent anticancer activity at a 4mg/ml concentration with inhibition percentages of 98.9 and 97.4% against HeLa and MCF7 cancer cell lines, respectively, while methanol extract at the same concentration showed potent activity against HeLa and MCF7 cancer cell lines with 97.2 and 95.6% inhibition percentage. From the previous results, the hexane fraction exerted a significant cytotoxic effect on both HeLa and MCF7 cells, with inhibition percentages reaching 99 and 92.4% at 4mg/ml of hexane fraction concentration, respectively. This was consistent with the cytotoxic effects of both terpenoids and steroids, which were found in the hexane fraction. Diosgenin, a naturally occurring steroid and triterpenoid found in some plants, has been shown to inhibit breast cancer [38, 39].

Conclusion

The results from this study on R. rothschildianus leaves indicated that the acetone extract fraction had significant potential in providing phytotherapy for diabetes and obesity, based on its potent inhibition of lipase, a-amylase, and a-glucosidase. In addition, the acetone fraction also showed a significant free radical scavenging activity. On the other hand, the hexane fraction showed significant inhibition of both HeLa and MCF7 cell lines, which might be related to its high content of terpenes and steroids. These observations in this study might lead to further in vivo studies to develop new natural pharmaceutical formulations effective in the treatment of obesity, diabetes mellitus, and some cancers.

This article is extracted from Jaradat et al. BMC Complementary Medicine and Therapies (2021) 21:107