INTRODUCTION:

Historically, natural products have been utilized for ancient times for the treatment of many illnesses and diseases. Natural product chemistry methodologies allow a broad collection of bioactive secondary metabolites from terrestrial and marine sources to be discovered.¹ thus natural products are considered a important source of drug progress upon different diseases.² According to the World Health Organization (WHO), medicinal plants would be the best origin to receive a variety of drugs. Approximately 80% of people from developed countries use traditional herbs which have medicinally active constituents obtained from plants.³

Conocarpus erectus L. is a perennial, evergreen shrub or tree height of up to 40 feet involving in Combretaceae family Consist of 20 genera and 300 species⁴. The conocarpus L. is genus, comprises of two different species C.lancifolius and C.erectus exist through the world such as southern Florida, Brazil and tropical West Africa. Commonly, C.erectus known as button mangrove or button wood.⁵ Leaves are simple spiral, opposite or whorled, lanceolate, petiolate, entire in long 2 to 10cm, Sometimes with a pair of petiolar glands, paniculate spikes and Inflorescence axillary as seen in figure 1⁶ Furthermore, the genus of this plant are reported to have numerous traditional uses as in anemia, tumors, bleeding, catarrh, diabetes, headache diarrhea, conjunctivitis, gonorrhea, syphilis, antipyretic and anti-inflammatory in the treatment of fever (Decoction of leaves) and swellings⁷ Its bark, as well as fruits, are utilized in the management of hemorrhoids, wounds.⁸ C.erectus L. is composed of a collection of constituents such as: primarily phenolic acids (gallic acid and ellagic acid, while minor phenolic acid one is 3,4,3-Trimethoxyellagic acid and Brevifolin carboxylic acid)⁹. Catechin, Rutin, quercetin, Apigenin, Myricetin, Syringetin, Quercetin-3-glycoside, are flavonoids that have been reported to isolated and characterized from the extract of C.erectus plant. ^,triterpenes, tannin and lignan.¹⁰ Pharmacologically, the plant reported to have been use as anticancer, antioxidant, antimicrobial and hepatoprotective activity.¹¹ Gallic acid with chemical name (3,4,5-trihydroxy benzoic acid) is a natural poly-phenolic compound located in Manufactured beverages such as green teas and red wines.^12,13 It is broadly distributed across the plant parts such as leaves (bearberry), bark and roots(pomegranates and gall nuts).^14,15 Gallic acid is a yellow-white crystals having a molecular weight of 170.12 g/mol, with melting point 250°C and solubility in water 1.1% at 20°C¹³. It can be synthesized via trihydroxycinnamic acid or caffeic acid from phenylalanine. Furthermore, It can be derived from 5dehydroshikimate directly an intermediate of the shikimate pathway.¹⁶ It is well used for its natural and potent antioxidative, anticarcinogenic, antiallergic, antimutagenic, anti-inflammatory, antibacterial, antiviral and anti arteriosclerosis activities.^17,18 It has also been found to induce apoptosis in some tumor cell lines and plays an essential role in the preventing of cancer growth and malignant transformation in vivo.^19,20,21 lupeol is a pentacyclic triterpenoid derived from lupane in which the hydrogen at the three beta position is replaced by a hydroxyl group. It occurs in the skin of lupin seeds. It is also located in numerous vegetables and fruits. Furthermore, which is have anti inflammatory and anti-cancer activity.^22,23

The main objective of this study was to isolate lupeol from hexane fraction and gallic acid from ethylacetate fraction and also investigate the effects of (hexane and ethyl acetate) fractions on viability of two types of human cancer cells (Human pancreas adenocarcinoma AsPC-1 and Human breast cancer MCF-7 by MTT assay.

MATERIAL AND METHODS:

General procedures:

IR spectra in KBR disk on Fourier-transform IR (FTIR) [Jasco-6100], high-performance thin-layer chromatography (HPTLC) analysis was carried out using CAMAG system (Switzerland), pre-coated silica gel GF254 (aluminum TLC) from Merck co., and the standard lupeol and gallic acid from Sigma-Aldrich chemicals Co. All the solvents were from Sigma-Aldrich chemicals Co and Merk chemical company. Human pancreas adenocarcinoma AsPC-1 and Human breast cancer MCF-7 were originally obtained from Sigma Aldrich and ATCC. The storage of it was in the Cell Bank of the Tissue culture Research Centre at the University of Al-Mustansiriyah/ College of pharmacy.

Plant material:

The Leaves of C.erectus plant were collected from the botanical home of AL-Mustansiriyah college of Pharmacy. Authentication of plant carried out by national herbarium center in Botany Directorate at Abu-Ghraib. The plants leaves were collected during the month of April (2019) and dried under shade conditions at room temperature during 12 days before grinding and weighing as a powder.

Preparation of leaves extracts:

The powdered leaves of C.erectus L. (100g) were defatted with hexane (1000mL). The defatted plant material was further extracted with methanol 85% (1200mL) using soxhlet extractor. The methanolic extract was concentrated by evaporation under reduced pressure using rotary evaporator. Then distilled water (35mL) was added to the methanolic extract, and the extract partitioned (3 time) with ethyl acetate (50mL) and allowed to settle over night. The lower aqueous layer was collected and labeled as fraction A, while the upper ethyl acetate layer was collected and labeled as fraction B.

TLC detection of triterpen and phenolic acid derivatives:

TLC analysis of hexane extract in comparison with standard lupeol was done by development with Chloroform: acetone (9:1) and Hexane: ethyl acetate (7:2) as a mobile phase. While TLC analysis of ethyl acetate fraction in comparison with standard gallic acid was done by development with chloroform: ethylacetate: acetic acid (5:5:0.2) and Ethyl acetate: methanol: water (15:1.25:1).

Isolation and purification of lupeol and gallic acid by preparative TLC:

For isolation of lupeol 1.54gm of hexane fraction was dissolved with hexane are subjected to preparative-glass plate using Hexane: ethyl acetate (7:2) mobile phase by glass pasture pipette 3-4 times on each plate while kept the spots should be drying between each application. The detection of separated spot was done by spraying side of plate with vanilline-sulphuric acid reagent. The band was scrapped off in comparison with standard, then eluted with hexane and full dried it by rotary evaporator. the purity of compound was checked by applying the separated compound on another preparative TLC and using Toluene : ethyl acetate :chloroform (5:1:4) solvent system to ensure the purity of separated compound.²⁴ For isolation of Gallic acid 3.32gm of ethyl acetate fraction was conducted on preparative TLC and it was developed in chloroform: ethyl acetate: acetic acid (5:5:0.2). The band was detected under UV light 254nm. Then eluted with methanol and acetone and full dried it by rotary evaporator. The Purification of compounds was checked by applying the separated compound on another preparative TLC.

Methods of identification of isolated compounds:

HPLC:

HPLC analysis was performed for identification of isolated compounds from hexane and ethyl acetate through comparison retention time of compounds with retention time of standards. The analyzed by (HLPC) method with UV-visible detection. The HPLC analysis was carried out by prominence HPLC system (SYKAM) and the separation was performed in a reversed phase (RP) ODS- C18 column (25cm x 4.6mm x5μm). The mixture was passed through a 0.45μm PVDF membrane and then 100μL of each sample was injected into the HPLC system. The separation was done for Hexane fraction by elution with isocratic mixtures, 30% methanol as solvent (A) and 70% acetonitrile as solvent (B)²⁴ A flow rate was set at 1ml/min for 10 minutes, detected by UV at 210nm.²² and for Ethyl acetate fraction to identify gallic acid by elution with isocratic mixtures 80% water as solvent A and 20% acetonitrile as solvent B, the PH 3 adjusted with ortho-phosphoric acid. A flow rate was set at 1ml/min for 10 minutes, detected by UV at 272nm.^25,26

HPTLC:

The HPTLC analysis was conducted to estimate isolated compounds from hexane and ethyl acetate extracts prepared with the same concentrations. The HPTLC analysis was performed by using HPTLC silica gel 60 GF 254s (10 x 20cm), the layer thickness was 0.5mm. The standards 3μL and the sample (3μL) were applied automatically on the plate by CAMAG Linomat 5. The plate was automatically submerged into anautomatic developing chamber (ADC2 CAMAG) using solvent system for gallic acid (toluene: ethylacetate: formic acid 36:12:5)²⁴ and for lupeol (hexane, ethyl acetate; 7: 2v/v)^27,28,with migration distance about 7.5cm. The plates were air-dried after development and scanned under UV (366 and 245nm) using CAMAG TLC scanner 4. The data were processed using winCATS software.²⁹

Chemical structure elucidation was obtained by FTIR and UV spectrophotometer:

Cytotoxic assay method:

Cell Viability by MTT Assay:

The MTT assay was used to analyise the effects of hexane and ethyl acetate fractions of conocarpus plant on the viability of pancreatic and breast cancer cells. A 100 µl from all cells suspensions (AsPC-1 and MCF-7) were dispensed into 96-well flat-bottom tissue culture plates at concentrations of 5 x 10³ cells\well and incubated for 24h under standard conditions,4x10³ cells per well incubated for 48h and 3 x 10³ cells per well incubated for 72h. After complete 24h, the cells were treated with (0.9, 1.8, 3.25, 7.5, 15 and 30µg/ml). following a recovery period 24h, 48h and 72h, the cell culture medium was removed and cultures were incubated with medium was contained 30μl solution of MTT (3 m\m MTT in PBS) (3-4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) at 37°C for 4hr. After the end of 4h this medium was removed by smooth inversion and tapping onto paper. The Control wells received only 100μl from growth media. Then 100 μl of dimethyl sulfoxide (DMSO) was added to each well and kept the plates were at room temperature in the dark conditions for about 15-20min. The measuring of absorbance of each well was by using multiscan reader instrument at a wavelength 540nm and the correction for background absorbance using 650nm wavelength.^30,31 The assay was performed in a triplicate and the percent of inhibition rate was calculated by:

% Inhibition rate = 100-%cell survival

Were A and B is the optical density for cisplatin (control) and optical density of test respectively.

Data Analysis:

All statistical analysis of hexane and ethylacetate fractions of MTT assay and IC₅₀ data on AsPC-1 and MCF-7 cells were performed by utilizing the nonlinear curve fitting software prism pad software. Comparison between all groups within the same plate of MTT was evaluated by one-way ANOVA with Turkey (prism pad software). Values of p < 0.05 were considered statistically significant.³²

RESULT AND DISCUSSIONS:

TLC detection of lupeol and gallic acid:

Hexane extract of the leaves was investigated by TLC which revealed the presence of lupeol through comparison the Rf value of the sample with Rf value of lupeol standards after spraying with vanillin-sulphuric acid spray reagent. on the other hand, For gallic acid comparison the Rf value of the spots of ethyl acetate fractions with Rf of Gallic acid standards and examined under UV light at 254nmas shown in figure 1 and table 1

Table 1: Rf values of phytoconstituents compared with standard of lupeol and gallic acid

Fraction type

Active compounds

Rf value of sample

Rf value of standard

Hexanfraction

Lupeol in Hexane: ethylacetate (7:2)

0.6

Ethyl acetate fraction

Gallic acid in chloroform: ethylacetae: acetic acid (5:5:0.2)

0.41

Figure 1: TLC detection of lupeol and gallic acid

Quantitative estimation of isolated lupeol and gallic acid:

The lupeol and gallic acid were isolated by PLC from C. erectus leaves and quantified about (0.65%) and (0.77%) respectively.

Identification of isolated triterpen and phenolic derivatives:

HPLC:

Isolated compound from hexane extract have retention time at 4.4min compared with 4.49min the retention time of standard lupeol as seen in figure 2A.

For gallic acid, isolated compound from ethyl acetate fraction have retention time at 3.86min compared with 3.88 min the retention time of standard gallic acid as seen in figure 2B.

HPTLC analysis for compounds isolated from ethyl acetate fraction:

The isolated compound from hexane and ethyl acetate fraction of Iraqi cultivated Conocarpus leaves was subjected to HPTLC along with standard lupeol and gallic acid developed under UV light at 254nm.

The result of HPTLC analysis showed that standard lupeol has maximum Rf value = 0.63 in compared with well- defined peak at maximum Rf = 0.63 of isolated lupeol as shown in Figure 2C.

The result of HPTLC analysis showed that standard Gallic acid has maximum Rf value = 0.13 in compared with well- defined peak at maximum Rf = 0.14 of isolated Gallic acid as shown in Figure 2D.

Structural elucidation of isolated lupeol by IR and UV spectrophotometer:

Structure elucidation by FT-IR for isolated compound showed the presence of broad stretching vibration of (OH) hydroxyl groups of aromatic rings at 3387-3333 cm-¹, and presence Stretching vibration of CH aliphatic (asymmetric and symmetric) bands at 2860. the IR specta data are listed in table. While UV spectrum show λ concentrated at 281nm.

Table 2: IR Bands with Interpretations of isolated lupeol.

Bands (cm־¹)	Interpretation
3387-3333	stretching vibration of OH
2862	Stretching vibration of C-H
1641	Stretching vibration of C=C
1450	C-H bending vibration
1031	Stretching vibration of C-O of 2º alcohol

Structure elucidation by FT-IR for isolated gallic acid showed the presence of hydroxyl groups of phenol at 3364 cm^-1 and of carboxylic acid at 3107 cm^-1, and presence bands at 1699 cm^-1 of carbonyl group as seen in table 3, while UV spectrum show λconcentrated at 272nm. The spectra data are agreement with previously reported literature for the gallic acid.²⁸

Table 3: IR Bands with Interpretations of isolated gallic acid.

Bands (cm^-1)	Interpretation of functional group
3489cm^-1	OH of phenol
3279cm^-1	OH of carboxylic acid; broad band at 3181cm^-1
1699 cm^-1	C=O of carboxylic acid
1514,1481 cm^-1	C=C aromatic ring stretching
1150-1300 cm^-1	C-O of phenol
873-671 cm^-1	C-H of aromatic ring; out of plane; strong

Percentage of cell death of MCF-7 cell line by hexane and ethyl acetate fractions:

To estimate the effect of both ethyl acetate and hexane fractions of Iraqi cultivated C. erectus L. on human breast MCF-7 cell line viability by using MTT assay in 96-well plates in comparison with the controlled anti-cancer drug (cisplatin) using different concentrations (30, 15, 7.5, 3.75, 1.87, 0.9, µg/ml) at different time 24, 48 and 72 hr.

Ethyl acetate fraction was significantly increased the MCF-7 cell lines death at 30 and 15mg/ml at 24, 48 and 72h p ˂ 0.005 versus other concentrations. While the results of Hexane fraction demonstrated that the percentage of MCF-7 cell lines death was less than Ethyl acetate fraction as shown in figure 3A p< 0.001.

Figure (6) represent ^{Dose-response curves of IC50
for}^{AsPC1 cells}when treated for 72hr with range of concentrations 30, 15, 7.5, 3.875, 1.87, 0.9 and 0.46µg/ml of ethyl acetate and hexane fractions by MTT assay. The results of IC50 for ethyl acetate fraction were (11.27µg/ml) and hexane were (14.23µg/ml) in AsPC1cell lines.

Cancer is the abnormal growth of cells in the body that can lead to death. It conceder the second cause of death in America and various countries.³³ Plants have confirmed to be an great natural source of anticancer remedy for many years. The chemo-preventive properties of phenolic compound (flavonoids and phenolic acids), triterpenes and hydroxylated coumarins detected in previous study are generally believed to reflect their ability to induce apoptosis.³⁴ Apoptosis is the induction process of programmed cell death. There are two pathways involved with in apoptosis one of them called intrinsic mitochondrial pathway in which lowering the level of Bcl2 and Bcl-xL protein family found in the mitochondria and The second pathway is extrinsic and named Fas/Fasl system in which the interaction of Fas/Fasl proteins on the target cell initiates the stimulation of numerous caspases cascade present intracellulary led to induce cell apoptosis.^35,36

However, from the overall illustrated results appeared the cytotoixic activity of the ethyl acetate fraction more than hexane extract of C.erectus leaves toward MCF-7 and AsPC-1cell lines.

The ethyl acetate fraction contains many polar to moderately polar compounds such as phenolic acid (gallic acid), flavanoids such as (kampferol, quercetin, and apigenen) and coumarin (umbelliferone) according to different analysis methods other study and all of these compound reported to have anti cancer activity³⁷. While from hexane fraction the non polar compounds triterpenes (lupeol) and steroids (β-sitosterol) were detected in the plant. In addition to some Phytochemical constituents (flavonoids and phenolic acids) were reported to be detected and isolated from C.erectus in another researches.¹⁰ In general, gallic acid give the important cause for cytotoxic activity of the ethyl acetate fraction have done in our work³⁸ Gallic acid possess cytotoxicity against MCF-7 cell line by induce apoptosis through both extrinsic and intrinsic pathways.³⁹ In contrast to, flavonoids and lupeol (triterpenes) isolated from different medicinal plants has been reported to exert their cytotoxic effect against MCF-7 and anther cell lines through intrinsic mitochondrial pathway.^40,41

Consequently, this can explain the results found by the study for the highest percentage of cell death that was observed in ethyl acetate extract in which it was more than hexane extract this due to the synergistic effect of many phenolic compounds presented in the ethyl acetate fraction.^42,43

CONCLUSION:

isolation of lupeol and gallic acid from C.erectus leaves growing in Iraq .Ethyl acetate fraction of Conocarpus leaves revealed high cytotoxic activity than hexane fraction when assessed their effects against human breast cancer cell line (MCF-7) and human pancreatic cancer cell line (ASPc-1).

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Received on 03.09.2020 Modified on 21.09.2020

Research J. Pharm. and Tech. 2021; 14(7):3495-3503.

DOI: 10.52711/0974-360X.2021.00606