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BY 4.0 license Open Access Published by De Gruyter Open Access July 2, 2022

Quantification of biomarkers and evaluation of antioxidant, anti-inflammatory, and cytotoxicity properties of Dodonaea viscosa grown in Saudi Arabia using HPTLC technique

  • Omer M. Almarfadi , Nasir A. Siddiqui , Abdelaaty A. Shahat , Ali S. Alqahtani , Perwez Alam , Fahd A. Nasr , Saad S. Alshahrani and Omar M. Noman EMAIL logo
From the journal Open Chemistry


Dodonaea viscosa (Sapindaceae) was collected from Riyadh, Saudi Arabia. For the simultaneous measurement of quercetin and kaempferol, a validated high-performance thin-layer chromatography (HPTLC) approach was devised in D. viscosa leaf extract. The antioxidant activity was evaluated using diphenyl 1-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assays. Moreover, the cytotoxic effect was tested against three cancer cell lines A549, HepG2, and MDA-MB-231. The potential anti-inflammatory properties of different fractions of D. viscosa were also evaluated using lipopolysaccharide (LPS)-induced THP-1 macrophages cells. The test samples include a crude extract of leaves and its solvent-soluble fractions of D. viscosa. The results showed that the crude extract and its fractions exhibited various significant biological activities, the fraction of chloroform demonstrated the highest free radical scavenging activity with IC50 values: 172.2 and 257.7 µg/mL for both DPPH and ABTS tests. Additionally, the chloroform fraction had the greatest cytotoxic activity against MDA-MB-231 (IC50 values: 24.6 ± 0.4 µg/mL). Moreover, the chloroform fraction exhibited the highest downregulation of the LPS-induced expression of TNF-α and IL-6. Quercetin and kaempferol were estimated concurrently in leaves crude extract using a validated technique on an HPTLC plate (10 cm2 × 10 cm2) with a combination of toluene–ethyl acetate–formic acid (5:4:0.2; v/v/v) as the mobile phase and a λ max of 254 nm. The amount of quercetin and kaempferol was found to be 31.8 and 15.01 mg/g of dried leaf extract, respectively. The presence of high levels of quercetin and kaempferol in D. viscosa leaves extract could explain its remarkable antioxidant, cytotoxic, and anti-inflammatory effects. The devolved HPTLC method can be used for routine analysis and standardization of D. viscosa crude plant material, extracts, and/or finished products using quercetin and kaempferol as appropriate markers.

1 Introduction

One of the primary causes of oxidative stress is an imbalance between oxidants and antioxidants in a biological system. This imbalance usually happens due to elevation in the production of reactive oxygen species (ROS) or impairment of endogenous antioxidant functions [1]. Although oxygen is important for a wide range of cellular activities like gene transcription and signal transduction; however, it turns coincidently into ROS such as superoxide radical, hydrogen peroxide, hydroxyl radical, and nitric monoxide radical anions which adversely affect the biological system [2,3]. These ROS trigger oxidative stress that damages the organelle’s structure and functions as well as causes various diseases like cancer, diabetes, neurodegenerative, and cerebrovascular [4,5,6]. Antioxidants are essential for scavenging ROS and maintaining oxidant/antioxidant equilibrium. Endogenous antioxidants and exogenous antioxidants are the two forms of antioxidants [1]. Endogenous ones are proteins exhibiting enzymatic or non-enzymatic activities such as superoxide dismutase and glutathione, respectively [7,8]. Exogenous ones such as vitamins, flavonoids, and phenols are derived from the plants as the main source. They also scavenge ROS during disequilibrium of the oxidant/antioxidant system preventing oxidative stress and eventually helping in providing a healthy biological system [9,10].

Dodonaea viscosa Jacq. (Family: Sapindaceae) is a perennial shrub growing in the tropical and subtropical regions. Australia is believed to be the center of origin of this species [11]. In Saudi Arabia, D. viscosa is extensively cultivated, notably in the Hijaz, Southern, and Eastern Border Provinces [12]. D. viscosa possesses a wide range of medicinal properties and has been used traditionally in a variety of ailments. It is used to treat sore throat, skin infections, and hemorrhoids [13]. It can be used to relieve swelling, inflammation, rheumatism itching, aches, and abdominal pain [14,15]. In Saudi Arabia, D. viscosa is commonly used for boils treatment [16]. Pharmacological studies have shown that D. viscosa extracts and their phytochemicals have antioxidant, anti-inflammatory, cytotoxicity, and antimicrobial properties [17,18,19].

According to the literature review, the results of phytochemical screening on this species demonstrated the existence of flavonoids, terpenoids, phenolics, alkaloids, tannins, and sugars [20]. Besides many other phytoconstituents, the earlier reports revealed the isolation and characterization of flavonoids such as rutin, quercetin and apigenin [21,22], viscosine [23], and viscosol [24]. The antioxidant activity of flavonoids has already been established in several studies. However, the free radical scavenging property was predominant in the protection of biological cells against ROS-mediated oxidative stress [17]. Structure–activity relationship studies demonstrated the antioxidant mechanisms of flavonoids which depend on the arrangement of functional groups in the flavan skeletal [25]. Hydroxy (OH) configuration on B-ring and the total number of OH groups on the flavan nucleus are the most determinant factors of scavenging ROS and reactive nitrogen species because they act as hydrogen donors that stabilizes these radical forming stable flavonoid radicals [26,27]. For that, flavonoids and flavonoid-containing plants possess antioxidant activity with fewer side effects and may play important role in the prevention of many oxidative stress-dependent diseases [28,29].

This study aims to evaluate antioxidant, anti-inflammatory, and cytotoxic effects of methanol extract and different organic solvent-soluble fractions of D. viscosa grown in the Eastern region of Saudi Arabia and quantification of its major flavonoids, quercetin, and kaempferol using a validated high-performance thin-layer chromatography (HPTLC) method.

2 Materials and methods

2.1 Plant material

Dodonaea viscosa Jacq. leaves (DVL) were collected from Riyadh, Saudi Arabia. The sample was identified and authenticated in the Department of Pharmacognosy, College of Pharmacy, King Saoud University, Riyadh, Kingdom of Saudi Arabia. A voucher specimen was deposited in the Herbarium of the department.

2.2 Chemicals and equipment

A rotary evaporator (BCHI Rotavapor R205, Swiss) and HPTLC (Eike Reich/CAMAG-Laboratory, Switzerland) were utilized. E. Merck Ltd, India, provided thin-layer chromatography (TLC) aluminum plates pre-coated with silica gel 60 F 254 (10 cm × 5 cm, 0.2 mm thick). All of the solvents and standards used were of analytical grade and came from the same place (purchased from Sigma-Aldrich).

2.3 Preparation of sample

About 300 g of air-dried leaves was crushed to a coarse powder, macerated in 1 liter of 80 percent methanol at room temperature for 2–3 days, and then repeated until the plant material was exhausted, yielding a crude methanol extract. Then, a rotary evaporator (45 rpm and 40°C) was used to filter and concentrate the crude methanol extract, yielding 111 g of dry methanol extract. After that, 100 g of this extract was suspended in water and fractionated by n-hexane, chloroform, and n-butanol, respectively, using 300 ml and repeated three times for each organic solvent. A rotary evaporator (45 rpm and 40°C) was used to concentrate the filtrates. The yields of the dried fractions for n-hexane, chloroform, and n-butanol solvents were 0.86, 27.56, and 23.95%, respectively. The dried portions were then collected into vials and kept at –20°C until needed.

2.4 Dodonaea viscosa antioxidant activity determination

2.4.1 Diphenyl 1-picrylhydrazyl (DPPH) assay

DPPH assay was used to screen the antioxidative effect of the total crude extract and organic solvent-soluble fractions of DVL, according to Brand-Williams et al. method with slight modifications [30]. In brief, 0.5 mL of each sample (2 mg/mL) was mixed with 125 µL DPPH reagent (1 mM) and 375 µL of spectroscopic methanol and incubated for 30 minutes. Finally, the anti-DPPH potential was demonstrated using ultraviolet (UV) spectrophotometer, absorbance measurement at 517 nm, according to the following formula:

% of anti-radical activity = { ( Absorbance of control Absorbance of sample/Absorbance of control ) } × 100 .

2.4.2 2,2′-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay

The antioxidant capacity of DVL extract and fractions was determined using the ABTS assay, which was modified slightly from that of Li et al. [31]. Briefly, aqueous solutions of ABTS (7 mM) and potassium persulfate (2.45 mM) were combined (1:1), incubated for 0.5 h, and maintained in the refrigerator for 24 h before being diluted with ethanol. Following that, different volumes of ABTS solution (50 µL) were combined with each sample and kept one hour in dark. Based on the reduction of ABTS, which was optically detected at 734, the antioxidant percentage activity of DVL extract and fractions was demonstrated using the formula below[32]:

% Of radical scavenging activity = { ( Abs control Abs sample/Abs control ) } × 100 .

% of radical scavenging activity = {(Abs control – Abs sample/Abs control)} × 100

2.5 Anti-inflammatory activity

Differentiation of THP-1 cells: The Human monocytic THP1 cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (Invitrogen), 2 mM l-glutamine, 1% antibiotics (penicillin and streptomycin), and kept it at 37°C in a humidified 5% CO2 atmosphere. Differentiation of THP-1 cells to macrophages was performed by treating the THP-1 cells with 5 ng/ml of PMA for 48 h. Once the cells get adhered, media was thrown, the adhered cells were washed twice with plane media, and then incubated in complete media for 12 h for obtaining resting macrophages. After 12 h of resting, cells were washed and incubated with and without the extracts for 1 h, then stimulated with 1 µg/mL of lipopolysaccharide (LPS), and then incubated for 24 h. After 24 h of incubation, cells were harvested by adding 1 mL of Trizol reagent (Invitrogen) to the adhered cells and kept at –80 until the time of processing.

2.6 Isolation of total RNA and reverse transcriptase-polymerase chain reaction (RT-PCR) for IL6 and TNF-α

After the treatment of macrophages with different fractions of extract and LPS, the expression of TNF-α and IL-6 was studied using semi-quantitative RT-PCR. For the isolation of RNA, the samples were thawed and RNA was isolated according to manufacturer instructions. In brief, chloroform was added and centrifuged at 13,000 rpm for 15 min. The colorless upper phase was gently collected in a separate Eppendorf, and isopropanol was added to precipitate the RNA, and it was centrifuged at 13,000 rpm at 4oC. The pellet was washed with 70% ethanol, and the pellet was air-dried at room temperature. The RNA pellet was dissolved in double-distilled water. The purity and concentration of RNA were determined using a nanodrop spectrophotometer. Superscript Vilo cDNA synthesis kit (Invitrogen) was used for the synthesis of cDNA by following the product information sheet as follows: 1 µg of total RNA, 4 µL of 5× Vilo reaction mix, 2 µL of 10× Superscript mix, and RNA-free water were used to make the total volume of 20 µL. The sample solution was incubated for 1 h at 42°C before being terminated for 2 min at 85oC. A semiquantitative PCR was carried out to determine the expression level of TNF-α and IL-6. The final volume (20 µL) of the RT-PCR mixture consists of 2 µL of cDNA, 4 µL of 5× FIRE pol Master mix (Solis Bio Dyne), and 10 pmols of each complementary primer specific for TNF-α forward primer 5′-CTGAAAGCATGATCCGGGAC-3′, reverse primer 5′-CGATCACTCCAAAGTGCAGC-3′, IL-6 forward primer 5′-AGGAGACTTGCCTGGTGAAA-3′, reverse primer 5′-CAGGGGTGGTTATTGCATCT-3′ and GAPDH forward primer 5′-GAGTCAACGGATTTGGTCGT-3′, reverse primer 5′ GACAAGCTTCCCGTTCTCAG-3′ as an internal control, and the rest of the volume was maintained by 4 µL RNase-free water. The sample was denatured for 5 min at 95oC, then amplified using 32 cycles of denaturation at 95oC for 30 s, annealing at 55oC for 60 s, and elongation at 72°C for 60 s, followed by final elongation at 72oC for 5 min. The 20 µL final amplification products were run on a 1.2% agarose gel stained with ethidium bromide and photographed using LICOR gel doc.

2.7 Cytotoxic activity ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) (MTT) assay)

MTT assay: MTT assay was performed to evaluate the potential cytotoxic activity (Nasr et al. [34]). Briefly, HepG2 (liver), A549 (lung), and MDA-MB-231(breast) cancer cells were plated in a 96-well culture plate at 5 × 104 cells per well and allowed to adherent for 24 h. Cells were then incubated with test extracts at different concentrations for 48 h, and doxorubicin was used as a positive control. Thereafter, 10 μL of MTT solution (5 mg/ml) was added to each well and incubated for a further 4 h. Acidified isopropanol (100 μl) was then added to solubilize the formazan, and the absorbance was determined using a plate reader (Bio-Tek, USA) at a wavelength of 570 nm. The half-maximal inhibitory concentrations (IC50) for each extract were calculated from the dose–responsive curve.

2.8 HPTLC analysis

2.8.1 Standard compounds

The biomarkers quercetin and kaempferol were purchased from T.C.I. (Figure 1).

Figure 1 
                     Structure of (a) quercetin and (b) kaempferol.
Figure 1

Structure of (a) quercetin and (b) kaempferol.

2.9 Development of HPTLC method for analysis of quercetin and kaempferol in methanolic extract of D. viscosa

On a 10 cm × 10 cm NP-HPTLC plate, quercetin and kaempferol concentrations in D. viscosa extract were measured (Merck, Germany). The researchers created a stock solution of quercetin and kaempferol (1 mg/mL) in methanol and diluted it with methanol to get seven distinct dilutions ranging from 20 to 140 µg/mL. All dilutions of kaempferol and quercetin as well as D. viscosa extract (12 μL of 1 g/mL concentration) were applied on an HPTLC plate through a microliter syringe connected with programmed TLC Sampler-4 (CAMAG, Switzerland) with a band size of 6 mm wide at a speed of 160 nL/s to provide a linearity range of 200–1,400 ng/band. After trial-and-error method, the mobile phase of toluene–ethyl acetate–formic acid (5:4:0.2; v/v/v) was found to be the most suitable with a clear and compact spot with no tailing. TLC plate was developed in a 10 cm × 10 cm pre-saturated twin-trough glass chamber (Automatic Development Chamber-2, CAMAG, Switzerland) at a specific temperature (25 ± 2°C) and specific humidity (60 ± 5%). The developed HPTLC was dried to give clear and compact spots of quercetin and kaempferol as well as different constituents of D. viscosa extracts and quantitatively analyzed at λ max = 254 nm in absorbance mode

3 Results

3.1 Antioxidant activity

The radical scavenging activity of D. viscosa extract and fractions are shown in Table 1 and Figure 2. In the DPPH and ABTS assays, D. viscosa exhibits a remarkable ability to scavenge free radicals at different concentrations. At a concentration of 1,000 g/mL, DVLCF had the highest antioxidant effect, with a value of 74.1% (Table 1). Similarly, DVLCF also had the highest antioxidant activity in (IC50 172.2 µg/ml and IC50 257.7 µg/ml). However, the antioxidant activity of the remaining crude extract and fractions were varied in both assays (Table 2).

Table 1

The scavenging ability of DVL extract and fractions in DPPH and ABTS assays

10 50 100 500 1,000
Radical scavenging activity in % (DPPH)
DVL-Crude 4.3 ± 2.4 12.4 ± 2.1 21.6 ± 2.5 37.9 ± 1.2 55.3 ± 1.7
DVL-Hex 5.2 ± 0.5 14.3 ± 2.1 23.2 ± 1.7 38.7 ± 2.2 56.7 ± 1.3
DVL-CHCl3 14.7 ± 2.1 25.3 ± 1.3 47.3 ± 1.9 61.3 ± 2.1 77.8 ± 2.2
DVL-ButOH 13.2 ± 3.6 23.1 ± 1.4 45.5 ± 1.3 56.5 ± 2.1 70. 6 ± 1.3
Ascorbic acid 83.7 ± 1.5 86.1 ± 1.3 87 ± 3.2 89.7 ± 2.4 91.7 ± 4.4
Radical cation scavenging activity in % (ABTS)
DVL-Crude 3.21 ± 1.2 12.2 ± 1.1 21.3 ± 2.1 37.3 ± 1.6 54.6 ± 1.7
DVL-Hex 3.3 ± 3.1 12.4 ± 2 21.6 ± 2.3 35.6 ± 1.1 54.7 ± 3.4
DVL-CHCl3 13.2 ± 3.3 23.3 ± 2.3 45.3 ± 1.2 57.3 ± 3.5 75.1 ± 2.3
DVL-ButOH 12.2 ± 1.2 20.2 ± 2.4 43.3 ± 2.2 54.3 ± 2.1 70.6 ± 1.2
Ascorbic acid 82.7 ± 2.4 84.2 ± 2.1 84.2 ± 3.2 86.2 ± 1.4 88.3 ± 2.6
Figure 2 
                  Sacavenging activity for (a) DPPH and (b) ABTS. Results are presented as mean ± standard deviation (SD).
Figure 2

Sacavenging activity for (a) DPPH and (b) ABTS. Results are presented as mean ± standard deviation (SD).

Table 2

IC50 of DVL extract and fractions

Extract IC50 DPPH (µg/mL) IC50 ABTS (µg/mL)
DVL-Crude 837.2 879.8
DVL-Hex 801.4 865.4
DVL-CHCl3 172.2 257.7
DVL-ButOH 250.9 350.7

3.2 Anti-inflammatory activity

3.2.1 Effect of extract on LPS-induced inflammatory gene expression

THP-1 differentiated macrophages were kept resting for 12 h (overnight) before adding the extract and incubating it for 1 h. After 1 h of incubation, the cells were treated with and without LPS for 24 h, followed by RNA extraction and cDNA synthesis. The semi-quantitative PCR results clearly indicate that LPS strongly upregulates the mRNA expression of TNF-α and IL-6 as compared to that of the negative control. Treatment of cells with different fractions reduced the LPS-induced expression of TNF-α and IL-6 with higher activity of chloroform fraction. There is no effect of crude extract on the LPS-induced TNF-α and IL-6 as shown in Figure 3.

Figure 3 
                     Effect of extract on LPS-induced gene expression of inflammatory cytokines. THP-1 differentiated macrophages were incubated with different fractions of extracts for 1 h and stimulated with 1 µg/mL LPS for 24 h. Expressions of TNF-α and IL6 were evaluated using semi-quantitative PCR. In this, a negative control was taken in the absence of LPS and extract whereas positive control was taken as only LPS treated. GAPDH was used as a loading control.
Figure 3

Effect of extract on LPS-induced gene expression of inflammatory cytokines. THP-1 differentiated macrophages were incubated with different fractions of extracts for 1 h and stimulated with 1 µg/mL LPS for 24 h. Expressions of TNF-α and IL6 were evaluated using semi-quantitative PCR. In this, a negative control was taken in the absence of LPS and extract whereas positive control was taken as only LPS treated. GAPDH was used as a loading control.

3.3 Cytotoxic activity

The cytotoxic activity of D. viscosa fractions was monitored using in vitro cytotoxic MTT assay. The results of D. viscosa cytotoxic activity against various cancer cells are summarized in (Table 3). All D. viscosa analyzed fractions revealed a dose-dependent anti-proliferative effect against all tested cancer cells (Figure 4). Among all fractions tested, it was observed that the D. viscosa chloroform fraction showed promising anticancer activity against breast (MDA-MB-231), lung (A549), and liver (HepG2) cancer cells with IC50 values 24.6, 50, and 52.6 µg/ml, respectively. The other D. viscosa fractions displayed a moderate inhibition toward a tested cancer cell line.

Table 3

Cytotoxic activity of D. viscosa fractions against various cancer cells

A549 HepG2 MDA-MB-231
Crude extract 134.6 ± 1.4 137 ± 1.3 47.3 ± 1.7
Hexane 50 ± 0.5 60 ± 1.2 36 ± 0.5
Chloroform 50 ± 0.8 52.6 ± 1.2 24.6 ± 0.4
Butanol 83.3 ± 1.7 94 ± 1.8 87.3 ± 1.6
Doxorubicin 0.8 ± 0.2 1 ± 0.3 0.9 ± 0.4
Figure 4 
                  Cytotoxic activity of D. viscosa fractions against various cancer cells.
Figure 4

Cytotoxic activity of D. viscosa fractions against various cancer cells.

3.4 Concurrent analysis of quercetin and kaempferol in methanolic extract of D. viscosa extract by HPTLC method

Many studies had conducted simultaneous determinations of flavonoids in various plants using validated HPTLC [33]. In this study, chromatographic conditions for the simultaneous detection of kaempferol and quercetin had been improved by using a modified mobile phase, toluene:ethyl acetate:formic acid (5:4:0.2; v/v/v). Sharp and well-resolved quercetin and kaempferol bands at Rf values (0.38) and (0.58), respectively (Figure 5a, Table 4). The fingerprinting data and 3D chromatogram displays of all tracks of rutin and D. viscosa methanolic extract (DVME) generated for extract and standards were visualized under UV and visible light (Figures 5b and 6), respectively. The plates were dried and screened at an absorbing wavelength (254 nm), and TLC scanner 4 was used to confirm the identity and purity of the quercetin and kaempferol bands in the plant samples. Line-to-line overlay spectra showed that the bands’ identity and purity matched those of the standard compound. As a result, there was no interference from any other component at the resolution site of quercetin and kaempferol. The quantities of quercetin and kaempferol in DVL extract were calculated to be 31.8 and 15.01 µg/mg, respectively.

Figure 5 
                  (a) Chromatogram of standards quercetin (Rf = 0.38), kaempferol (Rf = 0.58) (mobile phase: toluene: ethyl acetate: formic acid [5:4:0.2; v/v/v] at 254 nm); (b) chromatogram of estimation quercetin and kaempferol in D. viscosa extract.
Figure 5

(a) Chromatogram of standards quercetin (Rf = 0.38), kaempferol (Rf = 0.58) (mobile phase: toluene: ethyl acetate: formic acid [5:4:0.2; v/v/v] at 254 nm); (b) chromatogram of estimation quercetin and kaempferol in D. viscosa extract.

Table 4

Rf, linear regression data for the calibration curve of quercetin and kaempferol

Parameters Quercetin Kaempferol
Linearity range (ng/spot) 200–1,400 100–1,200
Regression equation y = 3.0318x + 48.448 y = 5.1719x + 445.41
Correlation coefficient (r 2) 0.997 0.993
Slope 3.0318 5.17
Intercept 48.448 445.41
Retention factor (Rf) value 0.38 0.58
Limit of detection (LOD) (ng) 142.14 120.83
Limit of quantitation LOQ (ng) 430.74 366.18
Figure 6 
                  Three-dimensional chromatogram displays all tracks of rutin and DVME.
Figure 6

Three-dimensional chromatogram displays all tracks of rutin and DVME.

According to the ICH guidelines, the method is accurate, repeatable, and precise (Table 5). The linearity ranges were found to be 200–1,400 ng/spot and 100–1,200 for each quercetin and kaempferol, respectively, while the correlation coefficient was 0.997 and 0.993 for each quercetin and kaempferol, respectively. For each standard chemical, the linear calibration curve was produced. The LOD values for both standards are 142.14 ng/spot and 120.83 ng/spot, respectively, whereas the limit of quantification (LOQ) values are 430.74 and 366.18 ng/spot, respectively. The average recovery was 98.9% for quercetin and 99.5% for kampferol (Table 6).

Table 5

Precision and accuracy of quercetin and kaempferol

Nominal Conc.1 Conc.2 Precision3 Accuracy4
Quercetin 200 198.8 ± 3.72 1.87 99
400 395.7 ± 5.60 1.42 99
600 596.8 ± 7.82 1.31 99.5
Kaempferol 150 147.3 ± 2.36 1.60 98
300 296.6 ± 4.21 1.42 99
600 597.9 ± 7.53 1.26 99
Quercetin 200 196.5 ± 3.55 1.81 98
400 394.8 ± 6.53 1.65 99
600 591.4 ± 8.15 1.38 99
Kaempferol 150 146.2 ± 2.12 1.45 97
300 294.4 ± 5.33 1.81 98
600 596.1 ± 10.47 1.76 98

1 Concentration in ng band-1; 2 Concentration (n = 6), 3 (CV, %) = [(Standard deviation)/(Concentration found)] × 100; Accuracy (%) = [(Concentration found)/(Nominal concentration)] × 100.

Table 6

Recovery studies of quercetin and kaempferol

Marker compound % Compound Theoretical concentration (ng/mL) Detected concentration (ng/mL) ± SD Recovery (%) RSD (%)
Quercetin 0 300
50 450 443.1 ± 8.12 98.4 1.83
100 600 593.8 ± 6.19 98.9 1.04
150 750 746.7 ± 7.86 99.6 1.05
Kaempferol 0 300
50 450 446.9 ± 6.31 99.3 1.41
100 600 597.1 ± 6.21 99.5 1.04
150 750 748.11 ± 8.78 99.7 1.17

HPTLC technique has become a valuable and reliable method for quality control of herbal drugs. It provides better phytoconstituents resolution and has high performance and cost-effectiveness. However, it is now widely used in the pharmaceutical and cosmetic sectors to assess the quality of both raw materials and finished products [34].

There are several real reasons for its increased popularity, including the need for fewer amounts of mobile phase and standard compounds, as well as a shorter analytical time. Furthermore, the products of this approach are renowned for their wide range of solvent systems, scanning wavelengths, and the ability to analyze numerous samples in a single run [35].

4 Discussion

Plants are rich in phytochemicals, which have a wide range of biological functions, including antioxidant and anticancer effects. Natural sources are constantly being explored in quest of biological agents with fewer side effects and better effectiveness than manufactured drugs. In this report, we evaluated the antioxidant, cytotoxic, and anti-inflammatory activities. In an in vitro investigation of the cytotoxicity of D. viscosa flowers, it was discovered that the flowers showed a cytotoxic effect against breast cancer [21]. Furthermore, the ethanol extract has been shown to have anticancer action by inducing apoptosis in a human breast cancer cell line [36]. These studies are in agreement with our data as we observed promising cytotoxic activities for the crude leave extract of D. viscosa and solvent-soluble fractions. The cytotoxic activity was determined against three types of cancer cells, A549 (lung) HepG2 (liver), and MDA-MB-231 (breast) cancer cells, using the MTT test. Crude extract and solvents-soluble fractions showed significant cytotoxic activity against all three cancer cell lines. On the other hand, regarding the antioxidant activity, D. viscosa growing in Yemen exhibited a strong antioxidant activity as reported by Mothana et al. [18], which confirms our results in which the antioxidant effect of the chloroform-soluble fraction of D. viscosa was the highest one (77.8%). While the crude extract showed the lowest antioxidant activity 55.3%, the n-butanol-soluble fraction was in between (70.6%). The antioxidant potential of fractions and crude extract of D. viscosa in this study was also in agreement with the radical inhibitory activity of Nephelium lappaceum L. (Sapindaceae), which was reported by López et al. [37]. It is almost presumed that plant antioxidant activity depends on the presence of polyphenols (e.g., flavonoids, tannins, and phenolic acids) and its capacity to donate hydrogen atoms or electrons and scavenge free radicals [38]. In general, ROS, e.g., hydrogen peroxide hydroxyl radical and superoxide anion radical, is involved in normal cell activity as well as pathological diseases, such as inflammation [39]. The role of ROS in inflammation models, e.g., carrageenan-induced pleurisy in rats, has been identified in numerous studies [40].

5 Conclusion

In our study, we observed that DVL exhibited potential antioxidant, anti-inflammatory, as well as cytotoxic effects against different cancer cell lines. Chloroform-soluble fraction proved to be a potent antioxidant, cytotoxicity, and anti-inflammatory activity; this supports the traditional uses of D. viscosa leaves. Simultaneous HPTLC quantification showed the concentration of quercetin is higher than kaempferol in DVL extract. In quality control, the HPTLC method can be used as an economic and simple technique for routine analysis and standardization of herbal drugs, extracts, and/or finished products using appropriate markers. Moreover, the findings of the present study revealed the significant cytotoxic, anti-inflammatory, and anti-oxidant potentials of plants recommend further work on isolation and testing of individual compounds for the above-mentioned effects.


The authors are thankful to the Researchers Supporting Project (RSP-2021/132), King Saud University, Riyadh, Saudi Arabia.

  1. Funding information: This research was funded by the Researchers Supporting Project (RSP-2021/132), King Saud University, Riyadh, Saudi Arabia.

  2. Author contributions: Conceptualization, A.S.A.; methodology, O.M.N., F.A.N. and O.M.A.; validation, P.A.; data curation, writing – original draft preparation, O.M.N., A.A.S., and S.S.A; writing – review and editing, N.A.S., O.M.N., A.S.A., and A.S.A., funding acquisition, A.S.A. All authors have read and agreed to the published version of the manuscript.

  3. Conflict of Interest: The authors declare that they do not have any conflicts of interest regarding the publication of this article.

  4. Ethical approval: The conducted research is not related to either human or animal use.

  5. Data availability statement: All the data related to these findings are included in the manuscript.


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Received: 2022-05-08
Revised: 2022-06-03
Accepted: 2022-06-07
Published Online: 2022-07-02

© 2022 Omer M. Almarfadi et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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