Spectral Characterization, Antioxidant And AntiTumor Activity of Kaempferol- 3-O-Alpha-L-Rhamnoside; A Flavonol Glycoside Isolated From The Ethyl Acetate Extract of Pithecellobium Dulce Leaves

Background: Pithecellobium dulce (Roxb.), an evergreen medium-sized, spiny tree which have vast nutritional values and widely used in ayurvedic medicines and home remedies. The plant has also been a rich source of biologically active compounds. The present study was designed to isolate pure compound and to know the ecacy as antioxidant as well as its anti-tumor activity on Ehrlich ascites carcinoma cell (EAC). Methods: The isolation of the compound was carried out by column chromatography and structure was revealed by 1 H-NMR and 13 C NMR. The antioxidant activity was investigated by the scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals as well as the inhibition of oxidative damage of pUC19 plasmid DNA, hemolysis and lipid peroxidation induced by 2, 2’-azobis (2-amidinopropane) dihydrochloride (AAPH) in human erythrocytes. In vivo anti-tumor activity of the compound was also evaluated by determining the viable tumor cell count, hematological proles of experimental mice along with observing morphological changes of EAC cells by uorescence microscope. Results: The isolated compound showed strong antioxidant activity in DPPH radical scavenging with IC 50 of 14.6μg/ml. It also effectively inhibited AAPH induced oxidation in DNA and human erythrocyte model and lipid per oxidation. In anti-tumor assay, 70.89±6.62% growth of inhibition of EAC was observed as compare to the control mice (p<0.05) at a dose 50mg/kg body weight. Conclusion: The a in preventing oxidative damage of biomolecules and the rst to evaluate the antioxidant activity kaempferol-3-O-alpha-L-rhamnoside a comprehensive employing a range of The present show pure antioxidant activity methods as cell growth inhibition on EAC cells. The study showed the DNA damage inhibition potential which could be used in cancer Further work be mechanism determine its


Background
Wide variety of chemical compounds synthesized by plants may have important biological functions with defend against attack from predators such as insects, fungi and herbivorous mammals. Many of these phytochemicals have bene cial effects on long-term health when consumed by humans and can be used to effectively treat human diseases. It is well known that the chemical compounds of plants affect the human body by the same process as the chemical compounds of conventional medicine thus herbal medicines do not differ greatly from conventional drugs in terms of how they work. This enables herbal medicines as effective as conventional medicines, but they are less likely to cause harmful side effects [1]. Various studies have reported that medicinal plants are the sources of many nutrient and non-nutrient molecules that could have antioxidant, anti-in ammatory, and antimicrobial activities [2].
Oxidative stress caused by free radical and reactive oxygen species is associated with various diseases.
A number of studies have been conducted worldwide to nd natural antioxidants in plant sources. Plants containing phenolic and avonoid compounds have been shown to have strong antioxidant activity.
Natural plant-derived products such as avonoids, terpenes, and alkaloids have received considerable attention in recent years due to their various pharmacological properties, including cytotoxic and cancer chemo protective effects [3].
About 50% of the drugs used for clinical trials were isolated from natural sources. DNA is present in every cells of body and human DNA remains continuously in exposure to free radicals attack that causes damage to DNA. Researchers all over the world are working on the aspect of relating the changes in DNA with evolutionary development [4][5][6]. Currently, there has been a growing interest in identifying free radical scavengers or antioxidants that prevent DNA from oxidative damage [7]. Another molecule, erythrocytes are considered the main target of free radical attack due to the high membrane concentration of polyunsaturated fatty acids [8]. Therefore, due to their sensitivity to oxidation, erythrocytes have been used as a cellular model to investigate oxidative damage in bio membranes.
The plant Pithecellobium dulce belongs to the fabaceae family and widely distributed in India, Huawei, and tropical Africa and especially along the coast. It has a long history of use in traditional system of medicine. The compound that was isolated from the leaves is a avonoid glycosides inhibits lipid peroxidation and cyclooxygenase (COX)-1 and COX-2. Several recent studies have indicated that the compound Kaempferol-3-O-alpha-L-rhamnoside inhibits the growth of breast cancer cells by stimulating apoptosis and that it is relatively non-toxic to normal cells [9]. Ehrlich ascites carcinoma (EAC) is a rapidly growing experimental tumor with very aggressive behavior and resembles human tumors [10]. The potent inhibitory effects of many isolated compounds and crude etracts of different medicinal plants against EAC have also been reported in literature [11]. However, the effects of this compound on the AAPH induced oxidative damage of erythrocytes and pUC19 DNA and also anti-tumor activity against EAC cell has not been investigated.
In this study, we aimed to investigate the effect of kaempferol-3-O-alpha-L-rhamnoside on antioxidant by employing various test models like DPPH scavenging, DNA and erythrocytes damage protection and to check the anti-tumor activity of this compound against EAC cell.

Collection of plant materials
The plant material was collected by following the ethics standard for research activity on plants established at Department of Pharmacy, University of Rajshahi, Bangladesh. Fresh samples of P. dulce leaves were collected from the relevant area of Kumarkhali, Kushtia during the months of November and December and were authenticated by a taxonomist, Prof. AHM Mahbubur Rahman, Dept. of Botany, University of Rajshahi, Bangladesh and given Accession no. 48085. A sample specimen was preserved in National herbarium Dhaka, Bangladesh.

Ethics of experimentation
The protocol for using human blood cells was approved by the Institutional Animal, Medical Ethics, Bio-Safety and Bio-Security Committee (IAMEBBC) for Experimentations on Animal, Human, Microbes, and Living Natural Sources at University of Rajshahi (Approval memo no-82/320/IAMEBBC/IBSc, 20 August, 2018). All procedures of this study adhere to the ARRIVE Guidelines for reporting animal research.

Extraction and fractionation
The collected healthy leaves were washed with tap water and then with distilled water and were spread out and dried at room temperature for about 15 -20 days and pulverized by mechanical grinder. 1.5kg of P. dulce leaves powder was rst extracted with n-hexane. The residue after n-hexane extraction soaked in 3.5 liter of methanol and kept it for 12 days with occasional shaking and stirring. The whole mixture was then ltered through cotton and then through Whatmann No.1 lters paper and was concentrated with a rotary evaporator under reduced pressure at 45°C to collect the crude methanolic extract (CME). Then sequentially fractionated towards polarity using the solvent n-Hexane, chloroform and ethyl acetate.The eluents of the four different polarity solvents were collected separately and concentrated by rotary evaporation under vacuum.

Isolation and characterization of the compound
In vitro biological assay, EAF had potent activity than other fraction and TLC assay also showed some distinct and prominent spot. Thus, later puri cation and isolation of active constituents were focused on EAF. This fraction was subjected a silica gel 60 column chromatography using n-hexane: ethyl acetate: methanol (1:2:1) and then ethyl acetate:n-hexane (95:5) as the eluent. Fractions 152 to170 was combined due to similar spot on TLC plate. Then fraction 152 to170 was further puri ed by preparative thin layer chromatography (PTLC) to isolate the target compound (1.2 g, Rf = 0.65). The compound was dissolved either in methanol and ethyl acetate depending on its solubility for analysis. The structure of the isolated compound was elucidated from the data obtained from 1 H-and 13 C-NMR spectra and by comparing with those reported in the literature [12]. The spectra of the pure compound were recorded on the Jeol-Ex at 400 MHz and 100 MHz and on FT NMR spectrometers using methanol as solvent.
Antioxidant activity DPPH radical scavenging activity The antioxidant activity of kaempferol-3-O-alpha-L-rhamnoside at various concentrations (3.125μg/ml to 50µg/ml) was evaluated via the DPPH radical scavenging system using BHT as standard. Brie y, 3ml of 0.04% DPPH in methanol was mixed with the compound solution, incubated in dark for 30 min and nally absorbance was measured at 517nm [13]. The experiment was repeated in triplicate manner and mean absorption was taken to calculate percentage of DPPH radical scavenging activity as follows- Where, A 0 -absorbance of blank, A 1 -absorbance of extract IC 50 (conc. in μg/ml) value required for 50% scavenging of DPPH was calculated from the graph plotted for the % of scavenging against concentration of the sample.

Antioxidant assay in human erythrocytes
Inhibition assay of AAPH-induced hemolysis Protective effect of the compound against oxidative damage of erythrocytes induced by AAPH was determined by the method described by Ribeiro et al [14]. Here, hemolysis was performed with AAPH as free radical initiator. An aliquot of 200 μl of 5% (v/v) erythrocytes suspension in PBS was mixed with 50 μl of compound with different concentrations (10, 25 and 50μg/ml in PBS pH 7.4). Standard ascorbic acid was used at concentration of 50μg/ml. To this, 0.5 ml of 50mM AAPH (dissolved in PBS) was added. The reaction mixture was incubated at 37 0 C for 6h in time dependent hemolysis. Each 1h interval sample was withdrawn, diluted with 2 ml PBS and centrifuged the mixture at 4000rpm for 10min.The supernatant portion was collected and absorbance was taken at 540nm. Reference values were determined using the same volume of erythrocytes in a hypotonic buffer (100% hemolysis) and kept under same condition as sample. The hemolysis percentage was calculated using the formula

Measurement of lipid peroxidation of erythrocytes
The effect of the compound kaempferol-3-O-alpha-L-rhamnoside on the inhibition of the production of malondialdehyde (MDA), a by-product of lipid peroxidation, was evaluated by incubation of a human erythrocyte suspension (5%) in 50 mM of the oxidizing agent AAPH [15]. Brie y a solution of three different concentration of the compound (10, 25 and 50μg/ml) was mixed separately with 0.5 ml of 50mM AAPH (dissolved in PBS). The reaction mixture was kept at 37°C for 6h for time dependent hemolysis. Each 2h interval one set of sample was withdrawn and 300μl H 3 PO 4 (0.44M), 750μl thiobarbituric acid (0.67%) were added to 1ml reaction mixture and incubated at 95°C for 1h. This was then cooled in an ice bath for 10min and 450μl trichloroacetic acid (20%) was added. The mixtures were centrifuged at 4000 rpm for 10min.The supernatant portion was collected and absorbance was taken at 532nm. A control solution of buffer with same volume of erythrocytes was kept under same condition as sample. MDA level was determined using molar extinction coe cient is 156mM -1 cm -1 at Beer Lambert Law and the value was expressed as pmol/g Hb.
MDA level = Antioxidant assay on plasmid DNA Protective effect on oxidative DNA damage Protective effect of the pure compound on DNA (pUC19) damage induced by AAPH was performed according to the Zhou et al [16]. The compound at the concentration (5, 10, 20, 40µg/ml) from a stock solution of 2mg/ml and 1µg DNA (2µl) were taken in eppendorff tubes after that 10µl AAPH was added to each mixture. The reaction mixture was adjusted to the total volume of 40µl by PBS and allowed to incubate for 30min at 37 0 C. After 30min incubation, 6X Determination of median lethal dose (LD 50 ) The median lethal dose (LD 50 ) value was determined by injecting the solution of the compound intraperitoneally in mice at various doses (5, 10, 25, 50, 100, 200mg/kg) and mortality at the end of the 24 h experiment was recorded. Doses were selected for this anti-tumor study by xed dose methods and were 25 and 50mg/kg [17].

Studies on in vivo EAC cell growth
The pure compound kaempferol-3-O-alpha-L-rhamnoside at doses of 25 and 50mg/kg per day were given to every mouse of group-II and III, respectively. Group -IV was treated with anticancer drug vincristine. Treatment was continued for 6 days and on seventh day after EAC cell inoculation, animals were sacri ced. EAC cells were collected by repeated washing with 0.9% saline and viable EAC cells per mouse of the treated groups were compared with untreated control [17]. Here, vincristine was used as standard at a dose of 6.25mg/kg/day. Vincristine is a chemotherapeutic drug that is poisonous to other cells.
When it is introduced into cells it binds to the cancer cell DNA which stops the cell division.

Morphological appearance of EAC cell
Morphological changes of EAC cells were examined by DAPI (4, 6-diamidino-2-phenylindole) staining after collecting the cells from non-treated EAC-bearing mice and mice treated with kaempferol-3-O-alpha-L-rhamnoside (25 and 50mg/kg/day) for 7 days. Then visual images were taken using uorescent microscope. Both uorescent and optical views were observed.

Studies on hematological parameters
To assess the haemotological parameters, Swiss Albinomice were divided into four groups (n = 5). All the animals were injected with EAC cells (0.1 ml of 1.6×10 6 cells/mouse) intraperitoneally except the normal group at the day zero. Group 1 served as the normal control, group II served as the untreated EAC control and group III served as the standard vincristine. Group IV and V were treated with compound kaempferol-3-O-alpha-L-rhamnoside at 25 and 50 mg/kg doses, respectively. On the 12th day, after tumor EAC cell inoculation, hematological parameters (Hemoglobin, RBC and WBC) were measured from freely owing tail vein blood of each mice of each group [18][19].

Statistical analysis
The data were analyzed by one-way ANOVA (analysis of variance) followed by multiple comparisons using Dunnett's post hoc and LSD test using SPSS software of 20version. All results were represented as mean ± standard deviation (SD). Differences at p < 0.05 level were considered to be statistically signi cant.

Results
Spectral characteristic 1 H-NMR and 13 C-NMR spectrum of the compound (Sz-02) are shown in Fig. 1 and On the basis of the 1 H NMR spectral features (Fig. 1) as well as 13 C NMR (Fig. 2) data, and comparison with the known kaempferol-3-O-alpha-L-rhamnoside [20], the compound was identi ed as kaempferol-3-Oalpha-L-rhamnoside.
Antioxidant activity DPPH radical scavenging activity The antioxidant activity of the compound evaluated by DPPH radical scavenging is shown Fig. 4   Morphological changes of EAC cells were examined by DAPI staining after collecting the cells from nontreated EAC-bearing mice and mice treated with kaempferol-3-O-alpha-L-rhamnoside (25 and 50mg/kg/day) after 7 days. EAC cells nuclei were round, regular, and homogeneously stained with DAPI in control group (solvent treated) as shown in Fig. 8. Apoptotic morphologic alterations such as membrane and nuclear condensation were noted in Kaempferol-3-O-alpha-L-rhamnoside treated EAC cells. These results indicated treatment with this compound could induce apoptosis in EAC cells.

Studies on hematological parameters
Hematological parameters of untreated EAC cell bearing mice on the day 12 were showed signi cant (P<0.05) changes when compared to normal mice ( Table 2). The total WBC count was found to increase with a reduction in the hemoglobin content and total RBC count. At the same time interval, treatment of compound (25 and 50 mg/kg) could brought back these altered parameters to normal values. The overall results of this study clearly demonstrated the anti-tumor activity of the compound against EAC.

Discussion
Pithecellobium dulce is used as astringent in dysentery, aborti cient, antidiabetic, anticonvulsant, antiulcer, larvicide dermatitis, eye in ammation, indigestion, intestinal disorder, ear ache, leprosy and tooth ache. Phytochemical investigation of bark and leaves had revealed the presence of β-sitosterol, saponin glycosides, oleanolic and echinocystic acids as sapogenins, echinocystic acid, bisdesmoside, dulcin, triterpenoids, acylated triterpenoid saponin, avanoids, saccharrides, long chain aliphatic hydrocarbons, and tannins [21][22]. In our study, kaempferol-3-O-alpha-L-rhamnoside; a avonoid that was isolated from leaves extract of P. dulce. To know its biological activity, we determined in vitro free radical scavenging and protection of oxidative damage on DNA and human erythrocytes as well as antitumor activity on EAC cells. The results of these experiments showed promising results. Antioxidants ght against free radicals and protect us from various ailments. Isolation and identi cation of many natural antioxidants from different plant materials have been studied. DPPH radical is one of the most common and stable chromogens used to estimate antioxidant activity of biological materials in a relatively short time. In our study, kaempferol-3-O-alpha-L-rhamnoside had potent DPPH radical scavenging activity and was higher than previously reported leaves extract of this plant [23]. For example, 28.9 and 83.2% DPPH inhibition for methanol and acetone (70%) of P. dulce leaves were reported at 100 µg/ml concentrations. But our compound showed 88.42 ± 0.49% DPPH scavenging activity at concentration of 50µg/ml and is more potent and e cacious than those crude extract of leaves.
For a better understanding of the antioxidant activity of the compound, we used a human erythrocyte to measure hemoglobin oxidation and lipid peroxidation inhibition. Free radicals attacking of erythrocyte membrane components (proteins and lipids) cause the alteration of membrane structure and function leading to hemolysis. The peroxy radicals generated from AAPH are capable of inducing lipid peroxidation and protein damage [23]. In the present study, the incubation of erythrocyte together with AAPH led to remarkable hemolysis that was consistent with previous ndings [24]. The present results clearly show that the pure compound has high capacity to prevent the oxidative damage induced in the erythrocyte membrane and to minimize the lipid peroxidation. Lipid peroxidation from AAPH induced radical results in the production of MDA which is responsible for cellular damage and associated with many of pathological events [25]. Since the compound showed ability to protect oxidation of erythrocyte membrane so the inhibition of lipid peroxidation was also expected and we found that the compound could inhibit the malondialdehyde formation in a dose dependent manner. Therefore, the protective effects of erythrocyte membrane and inhibition of MDA production of this pure compound may result from diminishing peroxyl radicals generated from AAPH during the incubation period as it was also effective in scavenging of DPPH free radicals.  (Fig. 7). This result demonstrated the DNA damage inhibition potential of this compound, therefore, can be used in cancer prevention.
So, next we investigated the role of this compound in cell growth inhibition on EAC bearing Swiss albino mice. The prolongation of the life span & reduction of tumor weight of cancer bearing mice is a very important and reliable criterion for judging the potency of any drug as anticancer agent. The effectiveness of the compound against EAC cell bearing mice has further been veri ed by monitoring the change in hematological and biological parameters. This study indicates that the number of cell growth decreased and number of apoptotic cells increased signi cantly at different doses. Previously reported the effect of kaempferol-3-O-alpha-L-rhamnoside on the viability of MCF-7 and HC-04 cells was evaluated. The treatment of cancer (MCF-7) and non-cancer (HC-04) cell lines with kaempferol-3-O-alpha-L-rhamnoside resulted in a dose-dependent inhibition of cell growth, [27] which also support our ndings. Anemia may occur during cancer chemotherapy. In our study, reduction in RBC or % in hemoglobin in tumor bearing mice may occur which is mainly due to iron de ciency or hemolytic or myelopathic conditions. Kaempferol-3-O-alpha-L-rhamnoside could signi cantly recover the hemoglobin content, RBC and WBC cell count that indicates the protective action of HM on the haemopoietic system. All these are measured are very important aspects in justifying the effectiveness of a compound in cancer chemotherapy. Figure 1 1H-NMR data of the isolated compound (Sz-02)   Effect of kaempferol-3-O-alpha-L-rhamnoside (Sz-02) and ascorbic acid on MDA formation in erythrocytes. Erythrocyte suspension at 5% hematocrit was incubated with 50 mmol/l AAPH at 370C in the absence or presence of compound or ascorbic acid at the indicated concentrations. Values are expressed as mean ± standard deviation.

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