Phytochemical Analysis, In-vitro Antiproliferative, Anti-oxidant Part 1

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Background: Rumex rothschildianus is the sole member of a unique section of the genus Rumex, in the family Polygonaceae. This species is a very rare small dioecious annual, endemic to Palestine that is traditionally used as food and for the treatment of various diseases. Therefore, the current investigation aimed to screen the chemical constituents, antioxidants, anti-a-amylase, anti-a-glucosidase, anticipate, and cytotoxic effects of four solvents fractions of R. rothschildianus leaves.

Methods: Dried powder of R. rothschildianus leaves was extracted in four solvents with different polarities. Several qualitative and quantitative phytochemical tests were performed to determine the components of the extracts. The colorimetric analysis was used for the quantitative determination of phenols, flavonoids, and tannins. In-vitro assays were performed to evaluate the extracts for antioxidant, anti-a-amylase, anti-a-glucosidase, and anticipate inhibitory activities, as well as cytotoxicity by MTS assay against cervical carcinoma cells line (HeLa) and breast cancer cell line (MCF7).

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Results: The acetone fraction of R. rothschildianus leaves showed the most significant antioxidant activity, due to having the highest content of flavonoids and phenolics， with an ICso value of 6.3±0.4 μg/ml， compared to 3.1±0.9 ug/ml for Trolox, and regarding lipase inhibition activity the acetone fraction showed the most potent activity with an ICso value of 26.3±0.6 μg/ml， in comparison with orlistat positive control ICso 12.3 μg/ml. The same extract was the most potent inhibitor of a-amylase and a-glucosidase， with ICso values of 19.1±0.7 μg/ml and 54.9±0.3 ug/ml， respectively， compared to 28.8， 37.1±0.3 μg/and of acarbose， respectively. The hexane fraction showed 99.9% inhibition of HeLa cells and 97.4% inhibition for MCF7 cells.

Conclusion: The acetone fraction of R. rothschildianus leaves might provide a source of bioactive compounds for the treatment of oxidative stress. Similarly, the hexane fraction indicates the promising antitumor potential of R. rothschildianus. Clearly, these initial indications need further purification of potentially active compounds, and ultimately, in-vivo studies to determine their effectiveness.

Keywords: Rumex rothschildianus, Antioxidant, Lipase, Amylase, Trolox, Phytochemistry, Anti-proliferative activity

Background

Plants have been used as therapies since ancient times. Roots, seeds, bark, leaves, and flowers have all been used for remedial purposes. In the present day, synthetic medicines are available and are effective in the treatment of a wide range of diseases; however, some people still prefer herbal medicines as they are viewed as being less harmful to the human body [1,2]. Medicinal plants are by definition the source of phytochemical compounds that possess therapeutic activities. These properties rely upon the presence of different secondary metabolites, such as phenolics, terpenoids, and alkaloids [3]. Rumex rothschildianus Aarons. is the sole member of a unique section of the genus Rumex, in the family Polygonaceae. This species is a very rare small dioeciously annual, endemic in Palestine. It has a mean height of 45 cm and is characterized by erect stems holding radical petiolate leaves, which are short-hastate at the base and short-acuminate at the apex. Flowers have a diameter of 3-4 mm, while pistillate flowers are about 2mm in diameter with a coriaceous membranous layer [4]. Rumex spp. are widespread in different regions of Turkey, and are represented by 22 species. Some of the most common species are R. patientia L, R. Crispus L, R. acetosa L. R. caucasicus rech, R. alpinus L. R. alpinus, and R. caucasicus are perennial plants distributed in middle and eastern Anatolia at an altitude of 1000-3000 m above sea level. The Rumex genus has been widely used in traditional medicine in Turkey to treat disorders, such as constipation, diarrhea, and eczema [5, 6]. The genus also has some laxative, diuretic, antipyretic, wound healing, and anti-inflammatory effects. Many people in the eastern part of Turkey use the young leaves of Rumex spp. as a preservative in cheese, as well as giving food aroma [7].

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A variety of research has been carried out on Rumex species, such as antimicrobial activities being reported for some species. Some bioactive phytochemicals have previously been found in Rumex vicarious L, such as carotenoids, tocopherols, polyphenols, flavonoids, and ascorbic acid, which have a role as antioxidants and natural detoxifying agents. The dietary intake of antioxidant phytochemicals, like carotenoids, phenolics, and flavonoids may protect against non-communicable diseases in humans, such as cancer, cardiovascular disorders, and other health problems related to oxidative stress [5, 8]. Harmful free radicals are known to play an important role in many major health problems, such as cancer, cardiovascular disease, rheumatoid arthritis, cataracts, Alzheimer's disease, and other degenerative diseases related to aging. Antioxidants are beneficial components that neutralize these free radicals before they can attack cells, and hence they prevent damage to cell proteins, lipids, and carbohydrates. A variety of both natural and synthetic antioxidants have been proposed for the treatment of human diseases. Such interest in the role of antioxidants in human health has prompted research in the fields of food science and medicinal herbs, assessing the function of herbs as antioxidants. Antioxidant action includes free radical scavenging capacity, inhibition of lipid peroxidation, metal ion chelating ability and also reducing capacity [9,10].

Cancer is one of the most global health care problems. The development and discovery of novel anticancer medication remain extremely important due to various factors. These factors include treatments that may cause major side effects or can be rather expensive. Alternatives that are safer biologically and more affordable are still highly desirable [11-14].

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Several plant species are considered potential sources of bioactive molecules such as atropine from Belladonna leaves, cocaine from coca leaves, vincristine from Vinca plant, and many others which still play an important role in modern medicine [15,16].

Useful therapeutic effects can come from mixing secondary products present in medicinal plants. These compounds are mostly secondary metabolites, like alkaloids, steroids, tannins, flavonoids, and phenolics, which are synthesized and deposited in specific parts of these plants [17,18]. The present study investigates the in-vitro anti-a-amylase, anti-α-glucosidase, anti-lipase, anti-proliferative and antioxidant activities of different fractions extracted from R. rothschildianus leaves.

Methods

Plant material, chemicals, and instruments

R. rothschildianus leaves were harvested from Western regions of Palestine, between February and March 2018. They were identified by Dr. Nidal Jaradat, from the Pharmacognosy Laboratory at An-Najah National University, under the voucher specimen code Pharm-PCT-2066. All chemicals were purchased from Sigma-Aldrich. A spectrophotometer-UV/Visible(Jenway°7135, Stafford-shire, UK), filter papers(Whitman No. 1, Washington, USA), shaker device (Memmert 531-25-1, Stockholm, Sweden), rotavap apparatus (Heidolph-VV 2000, Schwa-bach, Germany), grinder (Aero Plus 500 W Mixer Grinder, I01, Wan Chai, China), electronic-balance (Radwag, AS 220/c/2, Torunska, Poland), freeze dryer-BT85(Millrock Technology, China) and cryo-desiccator(Mill-rock technology, BT85, Kingston, USA)were used.

Preparation of extracts and fractionation

Dried powder of R. rothschildianus leaves was extracted by adding solvents sequentially based on their polarity, beginning with the non-polar solvent hexane, and then acetone (a polar aprotic organic solvent), methanol (polar alcohol), and finally distilled water(a polar protic solvent). For each extraction, about 25g of ground dried leaves were placed in 0.51 hexane for 72h in a shaker device at 100 rotations per minute at 25℃. Firstly, the hexane was replaced with 0.5L acetone, and then subsequently replacement involved equivalent volumes of methanol and water. Incubations in the solvents were as described above for hexane. Each organic fraction was filtered and concentrated under a vacuum on a rotary evaporator, while the aqueous fraction was dried using a freeze dryer. Finally, all crude fractions were stored at 4℃[19,20].

The yield of each fraction was calculated using the following formula:

Preliminary phytochemical assessment

Phytochemical screening tests of R. rothschildianus leaves in four fractions were carried out to identify active secondary metabolites. The qualitative results were expressed as (+) for the presence and (-) for the absence of bioactive phytochemicals[10,21].

Determination of total phenolic content (TPC)

The procedure to determine TPC was based on that of Cheung et al. TPC was expressed in milligrams of gallic acid equivalents per gram dry weight of leaves (mg GA/g dry weight). Freshly prepared 7.5% sodium carbonate solution was made by placing 7.5g Na2CO3 in a volumetric flask and adjusting the volume to 100ml with distilled water. A standard reference solution (gallic acid solution) was prepared by dissolving 100mg of gallic acid in distilled water to a final volume of 100ml. From this, a serial dilution was performed to obtain solutions of gallic acid at 100，70，50，40， and 10μg/ml）. The stock solutions of the fractions from leaves were prepared by dissolving 100mg of plant extract in distilled water, and adjusted to a total volume of 100ml. Reaction mixtures were prepared by mixing 0.5 ml of each fraction solution with 2.5 ml of 10% Folin-Ciocalteu's reagent, which was dissolved in water with 2.5 ml of 7.5% sodium bicarbonate. The sample tubes were incubated for 45 min at 45℃. Then, the absorbance of each was measured in a spectrophotometer at a wavelength of 765 nm. The working samples were prepared in triplicate for each analytic trial, from which the mean and standard deviation values were calculated [21].

Determination of total flavonoid content (TFC)

The TFC in the four R. rothschildianus leaf fractions was assessed using a calibration curve of rutin (standard reference compound). Results were expressed as milligram of rutin equivalent per gram dry weight of leaves extract (mg RU/g dry weight). A calibration curve for rutin was established using serial dilutions generated from a stock solution of 100 μg/ml. To make the stock solution， 10 mg of rutin was dissolved in 10ml of distilled water and then diluted to 100ml. Subsequently, the stock solution was diluted to provide rutin at concentrations of 10, 30, 40，50，70， and 100 μg/ml. For working solution preparation, 0.5ml of each fraction solution was mixed with 3 ml methanol, 0.2ml 10% AlCl3,0.2ml 1M potassium acetate, and 5ml distilled water, and then incubated at room temperature for 30 min. The previous steps were repeated for each of the fractions, after which, absorbance was measured at a wavelength of 415 nm. For a blank control, a working solution was set up with distilled water in place of the sample extract. The samples were prepared in triplicate for each analytic trial, from which the mean and standard deviation values were calculated [22].

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Determination of total tannin content (TTC) The protocol of Sun et al. was used to determine TTC in the four R. rothschildianus leaf fractions, being the most commonly used procedure. Catechin was used as a reference compound to construct a calibration curve. A 100μg/ml stock in methanol was prepared， from which a dilution series was generated to give catechin concentrations of 10， 30， 50，70， and 100 μg/ml. A 4% solution of vanillin in methanol was prepared freshly. Stock solutions of the fractions at 100 μg/ml were prepared using methanol as a solvent. For the working solution, 0.5ml of each fraction solution was mixed with 3ml vanillin solution and 1.5ml of concentrated HCl. The mixture was allowed to stand for 15 min, and then the absorbance at 500 nm was measured, using a working solution set up with methanol in place of the sample extract as a blank. All working samples were analyzed in triplicate, from which the mean and standard deviation values were calculated. Total tannin in each fraction was expressed in terms of catechin equivalents (mg of CAE/g dry weight of leaves)[23].

Antioxidant activity method

The free 2,2-diphenyl-picrylhydrazyl (DPPH) radical scavenging assay was used to measure antioxidant activity in the different fractions of R. rothschildianus leaves. A 1000μg/ml stock solution of each plant fraction was prepared in methanol. In addition， a 1000 μg/ml solution of Trolox was also prepared (the reference standard). A dilution series was prepared from the stock solutions for each fraction, giving six serial dilutions at 2, 5, 10,20, 50， and 100 μg/ml. One ml of each extract dilution was mixed with 1 ml 0.002 g/ml DPPH in methanol. One ml of methanol was added to give a final working volume of 3 ml. The DPPH solution was freshly prepared, as it was very sensitive to light. The blank control of the series concentrations was DPPH in methanol in a ratio of 1:2, without the addition of an extract. All working solutions were incubated at room temperature (25°C) in the dark for about 30 min. Optical densities were then measured with a spectrophotometer at a wavelength of 517 nm. The following equation was used to calculate % DPPH inhibition for each plant fraction, with Trolox as the standard compound:

where, Ag is the recorded absorbance of the blank solution, and Ats is the recorded absorbance of the tested sample solution [21].

Porcine pancreatic lipase inhibition assay

Stock solutions of 500 μg/ml were made from each plant fraction in 10% DMSO. From these, a dilution series of five concentrations of 50，100，200， 300， and 400 μg/ml were made. A 1 mg/ml stock solution of porcine pancreatic lipase in Tris-HCl buffer was prepared freshly just before use. The substrate, p-nitrophenyl butyrate (PNPB)was prepared by dissolving 20.9mg in 2ml acetonitrile. For each working solution, 0.1ml porcine pancreatic lipase was mixed with 0.2ml plant fraction from each member of the dilution series. Tris-HCl was added to make the final volume of the working solutions 1ml, and they were incubated at 37℃ C for 15 min. After incubation, 0.1 ml p-nitrophenyl butyrate solution was added to each test tube. The mixture was then incubated for a further 30min at 37℃. Pancreatic lipase activity was determined by measuring the hydrolysis of PNPB into p-nitrophenolate at 410 nm, using a UV spectrophotometer. The same procedure was repeated using orlistat as a standard reference compound. Percentage lipase inhibition by plant fractions was calculated with the following equation:

where, Ap is the recorded absorbance of the blank solution, and Ats is the recorded absorbance of the tested sample solution [24]. a-Amylase inhibitory assay

A100 mg of each fraction was dissolved in a few millimeters of 10% DMSO, and then further dissolved up to 100ml in 0.02 M Na HPOq/NaH-PO4,0.006M NaCl, pH 6.9 to give finally stock solutions with concentrations of 1000 μg/ml. From these， the following dilutions were prepared of 10,50,70,100, and 500 ug/ml, using 10%DMSO as the diluent. A 0.2ml volume of 2units/ml porcine pancreatic α-amylase was mixed with 0.2ml

plant fraction and was incubated for 10min at 30℃. After incubation, 0.2ml of a freshly prepared 1% starch solution in water was added, and the tubes were then incubated for at least three more minutes. At this point, the reaction was stopped by the addition of 0.2ml 3,5-dinitro salicylic acid (DNSA) color reagent and was diluted with 5ml of distilled water, before being heated at 90℃ for 10 min in a water bath. The mixture was then cooled to room temperature, and the absorbance was measured at 540 nm. The blank control was prepared using the same quantities described above, but replacing the plant fraction with 0.2ml buffer. Acarbose was used as a standard reference following the procedure described above. a-amylase inhibitory activity was calculated using the following equation: