Open Access

Anti-angiogenic activity of Middle East medicinal plants of the Lamiaceae family

  • Authors:
    • Qasem Abdallah
    • Ibrahim Al‑Deeb
    • Ammar Bader
    • Fayez  Hamam
    • Kamel Saleh
    • Amin Abdulmajid
  • View Affiliations

  • Published online on: June 12, 2018     https://doi.org/10.3892/mmr.2018.9155
  • Pages: 2441-2448
  • Copyright: © Abdallah et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Angiogenesis plays a crucial role in malignant tumor progression and development. The present study aimed to identify lead plants with selective anti-angiogenic properties. A total of 26 methanolic extracts obtained from 18 plants growing in Saudi Arabia and Jordan that belong to the Lamiaceae family were screened for their cytotoxic and anti-angiogenic activities using MTT and rat aortic ring assays, respectively. Four novel extracts of Thymbra capitata (L.) Cav., Phlomis viscosa Poir, Salvia samuelssonii Rech.f., and Premna resinosa (Hochst.) Schauer were identified for their selective anti-angiogenic effects. These extracts did not exhibit cytotoxic effects on human endothelial cells (EA.hy926) indicating the involvement of indirect anti-angiogenic mechanisms. The active extracts are potential candidates for further phytochemical and mechanistic studies.

Introduction

The cure for cancer remains elusive notably for the majority of the solid malignancies. Various challenges in treating cancers are linked to tumor progression and metastasis (1,2). The growth of solid malignances and their metastasis depends closely on adequate oxygen and nutrient supply, which ensures the formation of new blood vessels (angiogenesis) within the tumor that are vital for its growth (3). Therefore, anti-angiogenic agents may contribute in suppressing cancer growth by preventing nutrient and oxygen supply to the tumor. Plants have long been used as an important source of therapeutic agents against several diseases including cancer (48). While several natural products, such as taxanes and Vinca alkaloids are widely used in cancer treatment, the use of other plant products is limited to the support of the immune system and/or the increase in the anticancer effects of other anticancer drugs (4). The identification of plant extracts with anti-angiogenic activity and limited toxicity can potentiate the effects of currently used anticancer agents without increasing their side effects.

The targeting of tumor angiogenesis can be mediated via direct and indirect pathways (9,10). Direct anti-angiogenic agents exhibit their effects by inhibiting the growth and migration of endothelial cells. The targeting of endothelial cells within tumors by such direct anti-angiogenic agents may result in unwanted side effects due to their toxicity on endothelial cells found in other tissues. Indirect anti-angiogenic agents inhibit the neovascularization within solid tumors by suppressing the expression of proangiogenic factors, such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). The mechanism of indirect anti-angiogenic agents may also involve the growth inhibition of mural cells (pericytes) embedded within the vascular basement membrane of blood micro-vessels. This in turn leads to inhibition of the formation, stabilization, remodeling and function of blood vessels (11). Previous findings have shown the direct and indirect mechanisms of anti-angiogenic agents (1113).

In the present study, we screened 26 methanolic extracts of plants belonging to the Lamiaceae family for their cytotoxic and anti-angiogenic effects. Their mechanism of action with regard to their selective anti-angiogenic properties was further explored by evaluating their ability to inhibit the growth and migration of human endothelial cells.

Materials and methods

Chemicals and materials

Chemicals were purchased from Sigma-Aldrich; Merck KGaA, (Darmstadt, Germany). Cell culture media and supplements were obtained from Gibco; Thermo Fisher Scientific, Inc., (Waltham, MA, USA) and all sterile plastics used in cell culture and experiments were obtained from Corning Incorporated, (Corning, NY, USA) unless specified otherwise.

Cell lines and cell culture conditions

The human colorectal adenocarcinoma cell lines (HT-29, Caco-2 and DLD-1) were a kind gift from Dr A. Aljada (Department of Basic Medical Sciences, College of Medicine, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, ΚSA). The human endothelial cell line (EA.hy926) was obtained from the American Type Culture Collection (Manassas, VA, USA). The colorectal adenocarcinoma cell lines were routinely maintained in advanced RPMI-1640 medium (Reduced Serum Medium, cat no. 12633-012, Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 4 mM L-glutamine, 4% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin. EA.hy926 cells were routinely maintained in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 10% FBS and 100 U/ml penicillin-streptomycin. All the cells were propagated in humidified atmosphere at 37°C supplemented with 5% CO2.

Plant sample collection and extraction

A total of 18 plants belonging to the Lamiaceae family were collected from different locations in Saudi Arabia and Jordan and identified by analytical chemistry techniques. A voucher specimen of each plant was deposited to the Herbarium of Pharmacognosy Laboratory at the College of Pharmacy of the Umm Al-Qura University (Table I). The plants were air-dried in the shade at room temperature and then grinded finely. A total of 2 g of each plant were extracted with 20 ml methanol under sonication for 8 min and kept for 24 h at room temperature prior to filtration. The filtrates were dried in glass petri dishes under ventilation. Finally, the crude extracts were dissolved in Dimethylsulfoxide (DMSO) in order to produce a solution with a final concentration of 200 µg/ml. The extracts were kept at −20°C until further use.

Table I.

Methanolic extracts of the plants screened for cytotoxicity in MTT test and anti-angiogenic activity in rat aortic assay.

Table I.

Methanolic extracts of the plants screened for cytotoxicity in MTT test and anti-angiogenic activity in rat aortic assay.

Survival following 100 µg/ml exposure ±SD (%)Inhibition of micro-vessel formation (%)


No.Plants NamePart usedCodeHT-29Caco-2DlD-1Rat aortic ring
1Ballota undulata (Sieber ex Fresen.) Benth.APUQU-JO-3394.14±6.92106.28±6.5885.47±4.9942.67±1.78
2Eremostachys laciniata (L.) BungeAPUQU-JO-3998.55±3.24115.02±7.0999.43±1.8732.09±4.34
3Moluccella laevis L.APUQU-JO-2093.62±4.64104.8±1.3489.31±4.282.5±5.12
4Moluccella laevis L.ROUQU-JO-2073.88±2.52116.89±6.4178.59±1.81−78±8.64
5Phlomis kurdica Rech.f.APUQU-JO-2793.43±8.08109.95±3.4196.35±5.1635.76±8.24
6Phlomis syriaca Boiss.APUQU-JO-6954.48±0.5299.30±4.0372.27±1.8745.99±30.79
7Phlomis viscosa Poir.APUQU-JO-3168.35±15.6675.35±3.4764.94±5.3081.46±22.7
8Premna resinosa (Hochst.) SchauerSTUQU-SA-10956.26±2.72110.10±2.0282.02±4.9786.45±16.67
9Premna resinosa (Hochst.) SchauerLEUQU-SA-10922.90±1.53NDNDND
10Salvia aegyptiaca L.APUQU-SA-10715.34±2.79NDNDND
11Salvia ceratophylla L.APUQU-JO-4946.32±8.55NDNDND
12Salvia ceratophylla L.ROUQU-JO-4939.07±14.85NDNDND
13Salvia deserti Decne.APUQU-SA-11227.11±3.85NDNDND
14Salvia dominica L.APUQU-JO-429.58±2.78NDNDND
15Salvia hierosolymitana BoissROUQU-JO-28101.57±0.8198.04±3.0689.37±3.5544± 6.13
16Salvia hierosolymitana BoissAPUQU-JO-2898.68±5.24104.18±3.3084.56±4.17−25± 14.28
17Salvia multicaulis Vahl.APUQU-JO-4763.82±7.1195.64±4.2488.83±3.8146.24±5.39
18Salvia multicaulis Vahl.ROUQU-JO-4718.53±4.04NDNDND
19Salvia palaestina Benth.APUQU-JO-20982.00±2.7985.71±2.5297.80±2.58−12± 5.42
20Salvia samuelssonii Rech.f.APUQU-JO-4096.48±2.4097.08±3.1096.84±1.5355.37±12.93
21Salvia samuelssonii Rech.f.ROUQU-JO-4013.40±2.26NDNDND
22Salvia syriaca L.ROUQU-JO 4870.31±7.1388.89±6.3371.52±1.9557.43±10.85
23Teucrium polium L.APUQU-JO-2152.14±2.8091.46±5.1296.01±4.4594.95±7.14
24Teucrium polium L.ROUQU-JO-2116.52±5.13NDNDND
25Thymbra capitata (L.) Cav.APUQU-JO 2386.63±1.1483.95±2.6687.93±4.63100±0.0
26Thymbra capitata (L.) Cav.ROUQU-JO 2310.98±0.25NDNDND

[i] Viability of HT-29, Caco-2 and DLD-1 cells at 100 µg/ml of exposure is expressed as mean ± SD. The inhibition of the micro-vessel formation at 100 µg/ml is expressed as mean ± SD. Suramine was used at 100 µg/ml as a positive control and the mean percentage of inhibition was 63.39±2.41. SD, standard deviation, ND, not determined; AP, arial parts; RO, roots; ST, stems; LE, leaves; JO, Jordan; SA, Saudi Arabia.

In vitro cytotoxic assay on colorectal cancer cells

HT-29 cells (2,000 cells/well) were seeded in 96-well plates and left to adhere overnight at 37°C in a humidified incubator with 5% CO2. The cells were then exposed for 96 h to complete cell culture medium containing plant extracts (100 µg/ml). The final concentration of DMSO was 0.05% and each plate also contained blank wells, containing only medium and no cells, and control wells containing untreated cells. After the exposure time course, the MTT colorimetric assay was performed as previously described by Mosmann (14), with minor changes. Thus, the supernatant was replaced with 200 µl/well of cell culture medium containing 0.5 µg/ml MTT and incubated for 4 h. The insoluble formazan was dissolved by 150 µl DMSO per well and the absorbance was measured at 540 nm (Spectramax® plus 384; Molecular Devices, LLC, Sunnyvale, CA, USA). The average absorbance in the control wells corresponded to 100% survival and the cell viability percentage following 100 µg/ml of the plant extract exposure was determined. All the extracts that induced <50% growth inhibition at a concentration of 100 µg/ml, were considered as non-cytotoxic and were selected for the direct anti-angiogenic screening.

Rat aortic ring assay

The procedure was performed as described by Brown et al (15), with minor modifications. Female Wistar rats (12–14 weeks old) were obtained from the animal house facility of the College of Pharmacy of Taif University. The rats were housed under standard animal facility conditions and were provided free access to food and water. The rats were maintained in a 12-h light/dark cycle at 25±2°C with relative humidity of 50–60%. The in vivo protocol was in accordance with the animal welfare guidelines adopted by the Taif University and was approved by the Medical Ethics Committee of the Department of Pharmacology and Toxicology of the College of Pharmacy of Taif University. The rats were humanely euthanized by neck dislocation under anesthesia with ketamine/xylazine that was administered IP (70 µg/kg ketamine and 10 µg/ml xylazine). The thoracic aorta was harvested, cleaned from fibro-adipose tissue and residual blood clots, and placed into M199 medium in order to maintain its integrity. The thoracic aorta was cut into rings of 1 mm in length and each ring/well was explanted into a 48-well plate preloaded with M199 medium (300 µl/well) containing fibrinogen (3 µg/ml) and aprotinin (5 µg/ml). In order to form the lower layer, 10 µl of thrombin (50 U/ml in normal saline: bovine serum albumin) was added and the layer was left to solidify for 1.5 h at 37°C in a 5% CO2 supplemented humidified incubator. The upper layer M199 media (300 µl/well) consisted of 20% FBS, 2 mM L-glutamine, 0.1% (w/v) aminocaproic acid and 1% penicillin streptomycin and amphotericin B antibiotics (Lonza Group, Ltd., Basel, Switzerland). The plant extracts were added to the top layer at a concentration of 100 µg/ml, while suramine 100 µg/ml was used as a positive control. The concentration range of 50–25 µg/ml was used only for those extracts that inhibited the formation of the new vascularization at 100 µg/ml by 50% or more compared with the untreated control. The treatment of the extracts and the control samples was held in independent duplicates. Subsequently, the plates were restored into a humidified incubator (37°C supplemented with 5% CO2) for seven days. On day four, the media of the upper layer were changed with M199 media containing similar treatments and concentrations. After the completion of the treatment course, the formation of micro-vessels was evaluated using phase contrast microscopy following the method developed by Nicosia et al (16). The measurement of the distance of the micro-vessel outgrowth was analyzed using the ImageJ 1.51n® software (National Institute of Health, Bethesda, MA, USA).

The percentages of inhibition of micro-vessel formation were compared with non-treated control samples and were presented as mean ± standard deviation (SD). All the extracts that showed 50% or higher (≥50%) inhibition of micro-vessel formation at 100 µg/ml, were considered potentially active and were selected for further evaluation.

Scratch assay of endothelial EA.hy926 cells

This assay was used to evaluate the effectiveness of selected plant extracts to prevent the movement and growth of endothelial cells following induction of a scratch. The scratch was performed manually on a monolayer of cells as described by Liang et al (17). Subsequently, EA.hy926 cells (4×105 cells) were seeded into a 6-well plate. The cells were incubated in a 5% CO2 supplemented humidified incubator at 37°C for 48 h until they reached 90% confluency and/or higher. A straight line in the middle of the monolayer was scratched using a sterile 200 µl-pipette tip. The supernatant was discarded, and the monolayer was rinsed with PBS twice before the addition of extracts at concentrations of 100, 50 or 25 µg/ml in DMEM (2 ml). The control cells were treated with DMSO (0.05% in DMEM). A total of five images of different areas were taken for each scratch using an inverted light microscope at 12 and 24 h (Leica Microsystems GmbH, Wetzlar, Germany). The magnification used was ×50. The area of the scratch in each image was analyzed using ImageJ 1.51n (National Institute of Health, Bethesda) software. The resulted area of scratched monolayer at each time point was expressed as the percentage of inhibition of migration compared with the mean area of the scratch at 0 h. All experiments were performed in independent triplicates and the results were expressed as mean ± SD.

Growth inhibition assay on EA.hy926 cell line

EA.hy926 cells (1.5×103 cells/well) were seeded in a 96-well plate and left to adhere overnight at 37°C in a humidified incubator with 5% CO2. The cells were exposed to plant extracts at a different concentration range (200–12.5 µg/ml) for 96 h. The final concentration of DMSO was 0.1% or less and each plate also included blank wells, which contained only medium and no cells and control wells, which contained untreated cells. Following the completion of the exposure time course, the MTT colorimetric assay was performed as described above. The average absorbance in the control wells was considered as 100% survival and the IC50 values were defined as the extract concentrations that inhibited the cell growth by 50% after 96 h exposure. The results were obtained from three independent experiments and expressed as mean ± SD.

Statistical analysis

All data were analyzed using SPSS v.22.0 (IBM Corp., Armonk, NY, USA). The inhibition assay data were compared using one way ANOVA, and the significant differences were evaluated at a confidence interval of 95% and P<0.05. In addition, the Tukey post hoc statistical test was employed in order to produce pairwise comparisons among treatments.

Results

Cytotoxic activity

In the present study, 26 extracts were obtained from 18 plants of the Lamiaceae family and were evaluated for their antiproliferative activities against HT-29 colorectal cancer cells (Table I). The antiproliferative activity test of the extracts was performed with the aim to exclude those extracts with potential cytotoxic effects. Therefore, extracts which caused 50% growth inhibition or higher at a concentration of 100 µg/ml, were considered potentially cytotoxic. The non-cytotoxic effects of the selected 16 plant extracts were confirmed on two other colorectal cancer cell lines, and finally they were screened for their anti-angiogenic activity on rat aortic rings (Table I).

Antiangiogenetic activity

The extracts that caused higher than and/or equal to 50% inhibition in micro-vessel formation compared with untreated aortic rings were considered potentially antiangiogenic. The inhibition of micro-vessel formation in rat aortic rings was analyzed at a concentration of 100 µg/ml of extract (Table I). A total of six plants, namely Teucrium polium, Thymbra capitata, Phlomis viscosa Poir, Salvia samuelssonii, Salvia syriaca, and Premna resinosa were proven to have selective anti-angiogenic activity (Table I). These plants indicated no cytotoxic effects on cancer cells, although they significantly inhibited the formation of micro-vessels (Table I). The results of Teucrium polium and Salvia syriaca were in agreement with those demonstrated previously by Zihlif et al (18), with the exception of the Salvia syriaca, extract origin, which was derived from the roots, instead of the leaves. The remaining four plants were further investigated for their role in the inhibition of micro-vessel formation at lower concentrations (50 and 25 µg/ml) as shown in Fig. 1.

A significant drop in the percentage of inhibition was noted at lower concentrations, however no significant differences were observed among 50 and 25 µg/ml. Furthermore, the highest efficacy was noted for Thymbra capitata. The representative images of the effect of the selected plant extracts on rat aortic vessel outgrowth are shown in Fig. 2.

Antiproliferative and anti-migratory activities of plant extracts on human endothelial cells (EA.hy926)

The exposure of the endothelial EA.hy926 cells to the four selected plant extracts indicated no significant change in their growth rate (Fig. 3). Similarly, no significant effects were observed with regard to the endothelial proliferation and migration (Fig. 4). The latter results strongly indicated that these extracts did not mediate their anti-angiogenic effects via direct mechanisms of action. Selective anti-angiogenic assays on these crude extracts that can be conducted in future studies, can aid the evaluation of their therapeutic effects as complementary medicine and/or the isolation of their pharmacologically active ingredients.

Discussion

Tumor angiogenesis is linked to poor prognosis and metastasis (19). Co-administration of herbal medicines with anti-angiogenic properties as complementary treatment may enhance the therapeutic outcomes of anticancer therapy (20). Various Lamiaceae plants (e.g., Salvia and Phlomis species) have long been used in folk medicine for the treatment of various diseases including cancer (5,21). For example, the diterpenoids isolated from the Lamiaceae plants demonstrated a wide range of pharmacological activities including antibacterial, antifungal, cytotoxic, cardiovascular and insecticidal activities. Salvia triloba (Lamiaceae) has demonstrated direct and indirect anti-angiogenic activities by inhibiting human umbilical vein endothelial cells (HUVECs) proliferation and expression of VEGF (18,22). The AIS-L1023 extract from the leaves of Melissa officinalis (Lamiaceae) also inhibited tube formation of HUVECs by lowering the level of VEGF (23).

The micro-vessel formation assay in the aortic ring occurs by the proliferation and migration of endothelial cells, in addition to pericyte recruitment (24). Therefore, inhibition of angiogenesis in the cultured aortic rings could be mediated via direct and indirect mechanisms. The mechanism of action of the anti-angiogenic agents is mediated via direct and indirect pathways (25). The direct pathway involves the growth inhibition of endothelial cells via cytotoxic effects and/or via the inhibition of endothelial VEGF receptors. The indirect anti-angiogenic pathway, however, involves more complicated mechanisms and is active in the tissues surrounding the endothelial cells, thus leading to diminished expression of proangiogenic factors (26).

In conclusion, the present study identified several plants of the Lamiaceae family with potential selective anti-angiogenic activity. Their effects on angiogenesis were not mediated via a direct mechanism of action. Their activity could be attributed partially to the known compound carvacrol, which is the main constituent of the essential oil of Thymbra capitata (27). In addition, other constituents such as flavonoids could play a synergistic role in the anti-angiogenic activity of these extracts (28). With regard to the Salvia species, the diterpenes that are present in this extract have been reported to have anti-angiogenic effect, such as tanshinone, which is the main constituent of Salvia miltiorrhiza (29). Salvia samuelssonii could be a good target for further phytochemical studies as it has not been investigated with regard to its anti-angiogenic activity, with the exception of its essential oil, which has been characterized and includes phenylpropanoids, monoterpenes, sesquiterpenes and diterpenes (30). Phlomis viscosa is rich in bioactive compounds including oleanolic derivatives, iridoid glycosides, megastigmane, lignan glucosides, neolignan and phenylethanoid glycosides. The latter class of natural compounds includes verbascoside, isoacteoside, forsythoside B, myricoside, and samioside (31,32). To the best of our knowledge, this is the first study that examined Phlomis species with regard to its anti-angiogenic effect. Premna resinosa is growing in Saudi Arabia and was recently investigated for its cytotoxic and antimicrobial activities (33). A previous study revealed the presence of several flavonoids with considerable bioactivity in this plant species (33). The extracts of Salvia hierosolymitana roots, Phlomis syriaca aerial parts and Salvia multicaulis aerial parts have been reported to have 50% of inhibition of micro-vessel formation. The activity of Salvia multicaulis was attributed to the presence of diterpenes and flavonoids (34,35). The organs of the investigated plants have shown considerable differences in their activities, and this is attributed to the chemical diversity in the plant's organs. In some cases both aerial parts and roots have shown comparable effects, although they are phytochemically different, such as in the case of Salvia syriaca (36,37). Further studies are warranted to explore the possible indirect pathway of their anti-angiogenic mechanism and to identify their active constituents.

Acknowledgements

Not applicable.

Funding

The present study was funded by the Taif University (grant no. 1-436-4303) (for anti-angiogenic assays) and by the National Plan for Science, Technology and Innovation (MAARIFAH) King Abdulaziz City for Science and Technology, Kingdom of Saudi Arabia, award number (12-MED2310-10) through Science and Technology Unit (STU) at Umm Al-Qura University (for cytotoxicity assays).

Availability of data and materials

The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

Authors' contributions

QA planned, designed and conducted the present study. IA performed the cytotoxicity, rat aortic ring assay, scratch method and antiproliferative experiments. AM collected, identified and extracted the plants. KS and FH contributed in the scratch assay design and statistical analysis. AA contributed in rat aortic assay design, development and data analysis. All authors contributed to the scientific writing and the evaluation of the manuscript.

Ethics approval and consent to participate

The present study was approved by the Medical Ethics Committee at the Department of Pharmacology and Toxicology of the College of Pharmacy of Taif University.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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August-2018
Volume 18 Issue 2

Print ISSN: 1791-2997
Online ISSN:1791-3004

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Spandidos Publications style
Abdallah Q, Al‑Deeb I, Bader A, Hamam F, Saleh K and Abdulmajid A: Anti-angiogenic activity of Middle East medicinal plants of the Lamiaceae family. Mol Med Rep 18: 2441-2448, 2018
APA
Abdallah, Q., Al‑Deeb, I., Bader, A., Hamam, F., Saleh, K., & Abdulmajid, A. (2018). Anti-angiogenic activity of Middle East medicinal plants of the Lamiaceae family. Molecular Medicine Reports, 18, 2441-2448. https://doi.org/10.3892/mmr.2018.9155
MLA
Abdallah, Q., Al‑Deeb, I., Bader, A., Hamam, F., Saleh, K., Abdulmajid, A."Anti-angiogenic activity of Middle East medicinal plants of the Lamiaceae family". Molecular Medicine Reports 18.2 (2018): 2441-2448.
Chicago
Abdallah, Q., Al‑Deeb, I., Bader, A., Hamam, F., Saleh, K., Abdulmajid, A."Anti-angiogenic activity of Middle East medicinal plants of the Lamiaceae family". Molecular Medicine Reports 18, no. 2 (2018): 2441-2448. https://doi.org/10.3892/mmr.2018.9155