# In Vitro Antiproliferative and Antioxidant Effects of Extracts fromRubus caesius Leaves and Their Quality Evaluation

Authors: Daniel Mirosław Grochowski; Roman Paduch; Adrian Wiater; Adrianna Dudek; Małgorzata Pleszczyńska; Monika Tomczykowa; Sebastian Granica; Paulina Polak; Michał Tomczyk
Journal: Evidence-Based Complementary and Alternative Medicine
 (2016)

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## Abstract

The present study was performed to evaluate the effect of different extracts and subfractions fromRubus caesius leaves on two human colon cancer cell lines obtained from two stages of the disease progression lines HT29 and SW948. Tested samples inhibited the viability of cells, both HT29 and SW948 lines, in a concentration-dependent manner. The most active was the ethyl acetate fraction which, applied at the highest concentration (250 μg/mL), decreased the viability of cells (HT29 and SW948) below 66%. The extracts and subfractions were also investigated for antioxidant activities on DPPH and FRAP assays. All extracts, with the exception of water extract at a dose of 250 μg/mL, almost totally reduced DPPH. The highest Fe3+ ion reduction was shown for the diethyl and ethyl acetate fractions. It was more than 6.5 times higher (at a dose 250 μg/mL) as compared to the control. The LC-MS studies of the analysed preparations showed that all samples contain a wide variety of polyphenolics, among which ellagitannins turned out to be the main constituents with dominant ellagic acid, sanguiin H-6, and flavonol derivatives.


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## Body

## 1. Introduction

Drugs of natural origin have been used throughout history to cure or prevent diseases. Modern phytotherapy is engaged in the production of remedies from materials derived from plants and their use in effective and safe therapy. Their main action could be aimed at three aspects: cytostatic activity, especially when therapy concerns tumour tissue, and anti-inflammatory and antioxidative or free radical reduction actions. With all this in mind, we have tried to evaluate the cytotoxic and antioxidant activities ofRubus caesius extracts on two human colon cancer cell lines obtained from two stages of disease progression. Additionally, the full phytochemical profile of all the investigated extracts obtained fromR. caesius leaves based on the HPLC-DAD-MSn method has been characterized for the first time.R caesius is a well-known shrub (dewberry) extending from Europe to Siberia, but it can also be found in the United States. Folk medicine attributes many virtues toR. caesius. Further studies are required to confirm the pharmacological relevance of the findings, but now there are great expectations for its wide therapeutic application [1].


## 2. Materials and Methods

### 2.1. Plant Material and Preparation of Extracts and Their Fractions

The leaves from wild species ofR. caesius were collected during June-July 2012–2014 from Puszcza Knyszyńska, near Bialystok, Poland. A voucher specimen of plant RC-11027 has been deposited in the Herbarium of the Department of Pharmacognosy, Medical University of Białystok, Poland. All plant samples, extracts, and fractions were prepared according to previously described methods [2]. Yields are as follows: RC1, 83 mg; RC2, 79 mg; RC3, 101 mg; RC4, 9 mg; RC5, 28 mg; RC6, 96 mg.


### 2.2. HPLC-DAD-MS3 Analysis

The HPLC-DAD-MS3 analysis was performed using similar conditions described previously [2]. HPLC analyses of samples were carried out on a reversed-phase Kinetex XB-C18, 100 mm × 2.1 mm × 1.7 μm column (PHENOMENEX, USA). Compounds were analysed in negative and positive ion modes (the MS2 152, −162, and −176 amu). In the case of the detection of one of the neutral loss masses MS3 fragmentation was performed. Analysis was carried out using scan from m/z 70 to 2.200.


### 2.3. Cell Cultures

Two human colon tumour cell lines were used. HT29 (ATCC® HTB-38™) and SW948 (ATCC CCL-237™) cell lines representing early and late stages of tumour development were cultured as monolayers in 25 mL culture flasks (NUNC, Rochester, USA). All cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS (foetal bovine serum) (v/v) and antibiotics (100 U/mL penicillin, 100μg/mL streptomycin) (SIGMA, St. Louis, MO, USA) at 37°C in a humidified atmosphere with 5% CO2.


### 2.4. MTT Assay

The MTT assay is based on the conversion of a yellow tetrazolium salt by viable cells to purple crystals of formazan. The reaction is catalysed by mitochondrial succinyl dehydrogenase. Cell sensitivity toR. caesius extracts was analysed in a spectrophotometric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test according to Mosmann [3].


### 2.5. Neutral Red (NR) Uptake Assay

The NR cytotoxicity assay is based on the uptake and lysosomal accumulation of the supravital dye, Neutral Red. Dead or damaged cells do not take up the dye. The method was used as described earlier [4].


### 2.6. Nitric Oxide (NO) Measurement

Nitrate, a stable end product of NO, was determined in culture supernatants by a spectrophotometric method based on the Griess reaction. The course of the procedure has been described previously [5].


### 2.7. DPPH• Free Radical Scavenging Test

The free radical scavenging activity of extracts was analysed by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The test is based on the ability of antioxidants to reduce the stable dark violet radical DPPH• (SIGMA, USA) to the yellow diphenyl-picrylhydrazine. The methodology has been described in our previous study [5].


### 2.8. Ferric-Reducing Antioxidant Power (FRAP) Assay

The FRAP method was used to determine the antioxidative capacity of the tested extracts. The procedure has been described earlier [4].


### 2.9. Statistical Analysis

The biological experiments were repeated three times. The data were analysed using one-way ANOVA followed by Dunnett’s multiple comparison post hoc test. Only results with significance ofp≤0.05 were considered significant.


## 2.1. Plant Material and Preparation of Extracts and Their Fractions

The leaves from wild species ofR. caesius were collected during June-July 2012–2014 from Puszcza Knyszyńska, near Bialystok, Poland. A voucher specimen of plant RC-11027 has been deposited in the Herbarium of the Department of Pharmacognosy, Medical University of Białystok, Poland. All plant samples, extracts, and fractions were prepared according to previously described methods [2]. Yields are as follows: RC1, 83 mg; RC2, 79 mg; RC3, 101 mg; RC4, 9 mg; RC5, 28 mg; RC6, 96 mg.


## 2.2. HPLC-DAD-MS3 Analysis

The HPLC-DAD-MS3 analysis was performed using similar conditions described previously [2]. HPLC analyses of samples were carried out on a reversed-phase Kinetex XB-C18, 100 mm × 2.1 mm × 1.7 μm column (PHENOMENEX, USA). Compounds were analysed in negative and positive ion modes (the MS2 152, −162, and −176 amu). In the case of the detection of one of the neutral loss masses MS3 fragmentation was performed. Analysis was carried out using scan from m/z 70 to 2.200.


## 2.3. Cell Cultures

Two human colon tumour cell lines were used. HT29 (ATCC® HTB-38™) and SW948 (ATCC CCL-237™) cell lines representing early and late stages of tumour development were cultured as monolayers in 25 mL culture flasks (NUNC, Rochester, USA). All cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS (foetal bovine serum) (v/v) and antibiotics (100 U/mL penicillin, 100μg/mL streptomycin) (SIGMA, St. Louis, MO, USA) at 37°C in a humidified atmosphere with 5% CO2.


## 2.4. MTT Assay

The MTT assay is based on the conversion of a yellow tetrazolium salt by viable cells to purple crystals of formazan. The reaction is catalysed by mitochondrial succinyl dehydrogenase. Cell sensitivity toR. caesius extracts was analysed in a spectrophotometric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test according to Mosmann [3].


## 2.5. Neutral Red (NR) Uptake Assay

The NR cytotoxicity assay is based on the uptake and lysosomal accumulation of the supravital dye, Neutral Red. Dead or damaged cells do not take up the dye. The method was used as described earlier [4].


## 2.6. Nitric Oxide (NO) Measurement

Nitrate, a stable end product of NO, was determined in culture supernatants by a spectrophotometric method based on the Griess reaction. The course of the procedure has been described previously [5].


## 2.7. DPPH• Free Radical Scavenging Test

The free radical scavenging activity of extracts was analysed by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The test is based on the ability of antioxidants to reduce the stable dark violet radical DPPH• (SIGMA, USA) to the yellow diphenyl-picrylhydrazine. The methodology has been described in our previous study [5].


## 2.8. Ferric-Reducing Antioxidant Power (FRAP) Assay

The FRAP method was used to determine the antioxidative capacity of the tested extracts. The procedure has been described earlier [4].


## 2.9. Statistical Analysis

The biological experiments were repeated three times. The data were analysed using one-way ANOVA followed by Dunnett’s multiple comparison post hoc test. Only results with significance ofp≤0.05 were considered significant.


## 3. Results and Discussion

Many species classified to the genusRubus have been recognized as potential agents with significant effects on human health [6–10]. In the present work we selected leaves of blackberryR. caesius (dewberry) species traditionally used as a remedy to treat many diseases, among them gastrointestinal bleeding and diarrhoea [1, 11]. More recently, Dudzińska and coauthors indicated that the extracts obtained from dewberry leaves demonstrate antiplatelet activities in whole blood, where neutrophils play a pivotal role in mediating their effects on platelets. Although these extracts do not hamper the neutrophil oxidative metabolism and do not influence the expression of neutrophil adhesive receptors, they demonstrate an ability to lower the reactive oxygen level produced by neutrophils [12]. According to the reviewed literature, little is known about the potential antiproliferative and antioxidant activity of dewberry’s leaves which encouraged us to investigate this plant growing in Poland. In addition, there is no solid evidence describing the chemical composition of the species.For the first time, we initiated a detailed phytochemical analysis of secondary metabolites and confirmed the presence of derivatives of quercetin and kaempferol, as well as ellagitannins [1, 11]. The fingerprints of the analysedR. caesius extracts were established using the HPLC-DAD-MS3 method. The analysis revealed the presence of thirty-five constituents (Figure 1) comprising ellagitannins and their derivatives, phenolic acids, as well as flavonoids. In the RC1 (water), RC2 (50% methanol), and RC3 (methanol) extracts ellagic acid [22] and sanguiin H-6 [23] were detected as the dominating constituents. The subfractions RC4 (diethyl ether), RC5 (ethyl acetate), and RC6 (n-butanol) contained a wide variety of phenolic acids [2,5,9,11,12,16,22,31and32], flavonoids, mainly, quercetin [18,19,26 and34], and kaempferol [25,27,30,33 and35] derivatives, as well as ellagitannins and related compounds [4,6–8,14 and29]. Table 1 contains detailed UV-Vis and MS data for all the detected compounds together with their preliminary or full identification. These phytoconstituents express reductive activity on free radicals and may limit the appearance of mutations or even participate in DNA repair [24]. There are a few reports concerning the antitumour activity of otherRubus leaves extracts, but no data are available supporting the extracts from dewberry leaves [15, 16, 22, 25, 26]. Previous studies on in vitro models suggest that berry fromRubus species may influence colorectal cancer cell survival in concert terms proliferation and apoptosis [17]. Komes and coworkers also revealed that infusion fromR. fruticosus leaves may induce cytotoxic action against human colon cells, depending of time and concentration [26]. On the other hand, cancer development is closely associated with inflammation and mutatory microenvironments containing free radicals. In another study, a triterpenoid-rich fraction fromR. coreanus has been shown to express strong anti-inflammatory activity towards injured colonic tissue [27].Table 1
MS and UV-Vis data of compounds detected in extracts and fractions prepared from leaves ofR. caesius.
Number Compound name Retention time [min] UV [nm] [M − H]−  m/z MS2 ions MS3 ions [M + H]+  m/z MS2 ions MS3 ions NL detected [amu] (1) Gallicacids 3.3 265 169 125b — 171 — — — (2) Unknown phenolic acid 3.4 229, 314 183 139b — 185 — — — (3) Unknown compound 7.2 214, 260, 292 363 329, 325, 278, 153b — 365 333b, 289, 269 — — (4) Unknown ellagitannin 10.2 260 391 a/783 481, 301b, 377, 311 — 802 c 785, 767, 483, 465, 303, 277b — — (5) Methylgallates 13.9 271 183 125b — 185 153b — — (6) Galloyl-HHDP-glucose isomer 15.6 260 633 481, 463, 343, 301b, 275 301b, 275 652 c 482, 465b, 447, 321, 303, 277 — 152 (7) Unknown ellagitannin 16.3 263 391 a/783 481, 301b, 377, 311 — 802 c 785, 767, 483, 465, 303, 277b — — (8) Galloyl-HHDP-glucose isomer 16.8 261 633 481, 463, 343, 301b, 275 301b, 275 652 c 482, 465b, 447, 321, 303, 277 — 152 (9) Unknown phenolic acid 18.3 237, 301sh, 321 179 135b — 181 — — — (10) Unknown ellagitannin 23.4 260 459 — — 461 — — — (11) p-Coumaric acid hexosidet 27.2 247, 300sh, 325 325 314, 163b, 259 — 355 233, 161b, 135 — — (12) Methylbrevifolincarboxylates 29.9 211, 277, 356 305 273b, 245 — 307 293, 275, 247b, 219 — — (13) Unknown compound 30.2 217, 252, 274, 337 330 327, 287b, 269, 214 — 332 314, 289b, 227 — — (14) Unknown ellagitannin 32.2 261 783 a/1567 1265, 1103, 933, 633, 301b — 1591 b — — — (15) Unknown phenolic acid 32.5 218, 310 279 272, 190, 163b — 281 147b — — (16) Ellagic acid derivative 33.7 250, 358 331 287b — 333 315, 289b, 272 — — (17) Unknown ellagitannin 34.1 262 783 a/1567 1265, 1103, 933, 633, 301b — — — — — (18) Quercetinrhamnoglucuronidet 34.4 255, 263sh, 351 623 321, 301b — 625 479b, 449, 303 303b 146 (19) Quercetinpentosoglucuronidet 35.1 256, 264sh, 351 609 429, 301b, 285 — 611 479b, 449, 303 461, 303b 132 (20) SanguniinH-2t 35.5 263 551 a/1103 935, 633, 541, 469, 301, 169 — — — — — (21) Ellagic acid pentoside 36.3 251, 354 433 387,301b, 161 257b 435 417, 303b, 219 285b 162 (22) Ellagicacids 37.0 250, 353 301 273, 257b, 245, 187 — 303 295, 285b, 257, 207, 147 — — (23) Sanguniin H-6t 37.4 260 934 a/1870 — — — — — — (24) Unknown compound 37.5 255, 350 431 269b — 433 271b, 174 — — (25) Kaempferolrhamnoglucuronidet 38.1 265, 347 607 321, 285b, 257 — 609 463b, 447, 287, 307, 231 447, 287b 146 (26) Quercetin 3-O-glucuronides 38.8 255, 263sh, 351 477 431,301b, 179, 151 273, 257, 179b, 151 479 303b — 176 (27) Kaempferolpentosoglucuronidet 39.3 265, 343 593 307, 285b — 595 549, 465b, 353, 287 445, 329, 287b 132 (28) Unknown compound 40.1 251, 324 309 277, 179, 161b — 311 — — — (29) Unknown ellagitannin 42.3 260 558 a/1117 935, 633b, 483, 459, 301 — (30) Kaempferol 3-O-glucuronides 43.1 263, 341 461 415, 285b, 257, 175 267, 257b, 241, 229 463 445, 371,287b, 203 258, 241b, 213, 173, 121 176 (31) Unknown phenolic acid 45.8 230sh, 294sh, 328 359 249, 223, 197, 179, 161b — 361 163b — — (32) Unknown phenolic acid 47.0 235, 298sh, 325 503 485,341b, 281, 251, 221, 179 323, 281, 271b, 221, 179 504 — — 162 (33) T i l i r o s i d e s 58.5 266, 295sh, 315, 353sh 593 447, 307, 285b 327, 285b, 255 595 329, 309b, 287, 235, 217, 165 — 146 (34) Quercetin derivative 59.9 255, 265sh, 353 533 515, 357, 301b — 535 303b, 215 — — (35) Kaempferol derivative 63.3 263, 343 663 645, 499, 399, 285b — 665 519b, 379, 287 287b, 159 146 ⁢M-2H2-a, b[M+Na+, M+H2Oc+, tentativet assignment, comparisonss with chemical standard have been made, b: base peak (the most abundant ion in recorded spectrum), and in bold: ions subjected to MS3 fragmentation.Figure 1
UHPLC chromatograms ofR. caesius samples (RC1–RC6) recorded at 254 nm.Therefore, we decided to aim our study at the cytotoxic and reduction activity ofR. caesius leaves in human colon carcinoma cells. Studies on the biological activity of different extracts and subfractions obtained from dewberry were based on two analyses (MTT and NR assays) which were performed on two human colon tumour cell lines HT29 (Duke’s A) and SW948 (Duke’s C). They were selected to show the reactivity of the early stage of this tumour development. Our study revealed that the testedR. caesius extracts expressed no cytotoxic activity. Samples RC2, RC5, and RC6 in a range of concentrations up to 200 μg/mL induced mitochondrial action about 20% above the HT29 cells control (Figure 2). The SW948 cell line was more sensitive to the activity of the tested samples (RC1–3, RC5) than the HT29 line. The inhibitory effect was concentration-dependent. On the other hand, samples RC4 and RC6 induced succinyl dehydrogenase activity. The tested extracts inhibited, in a concentration-dependent manner, the viability of cells of both HT29 and SW948 lines. The most active was RC5, which, applied at the highest concentration (250 μg/mL), decreased the viability of cells (HT29 and SW948) below 66% (Figure 3). The butanolic fraction (RC6), which at the highest concentration did not decrease the viability of cells below 87%, was less active. Our tests revealed thatR. caesius extracts possess reductive activity. All extracts, with the exception of RC1 at a dose of 250 μg/mL, almost totally reduced DPPH (Figure 4). RC1 at this concentration reduced only half of the radical. Fractions RC4 and RC5 at the lowest concentrations used expressed strong antioxidative activity. IC50 values of the tested samples’ activity and their comparison to the Trolox action are presented in Table 2. Antioxidant activity of the selected extracts was also determined by the FRAP method, which is based on the analysis of the Fe3+ ions reduction ability of the tested compounds. The highest Fe3+ ion reduction was shown for the RC4 and RC5 extracts. It was more than 6.5 times higher (at a dose 250 μg/mL) as compared to the control (Figure 5). This result is comparable to 155 μg/mL of ascorbic acid reductive activity. Similarly to DPPH, the lowest reductive action was shown for the RC1 extract. At the highest concentration applied, its activity was only 2.2 times (activity corresponding to 53 μg/mL of ascorbic acid) stronger than the control.Table 2
DPPH radical reduction assay. IC50 values of extracts action compared to corresponding activity of synthetic vitamin E derivative (Trolox).
Samples IC50 of extract activity (μg/mL) Trolox concentration (μg/mL) which corresponds to IC50 of extract activity RC1 240.9 20.5 RC2 101.8 26.8 RC3 123.5 32.7 RC4 54.8 23.0 RC5 44.5 21.3 RC6 131.3 23.0Figure 2
The effect ofRubus caesius extracts influence on HT29 (a) and SW948 (b) metabolic activity. The study was conducted for 24 h. The MTT assay. The results are shown as a percentage of the control, arbitrarily set to 100%.
(a) (b)Figure 3
The effect ofRubus caesius extracts influence on HT29 (a) and SW948 (b) cellular membranes stability. The study was conducted for 24 h. The NR uptake assay. The results are shown as a percentage of the control, arbitrarily set to 100%.
(a) (b)Figure 4
The DPPH free radical scavenging activity. The % of reduced radicals byRubus caesius extracts is compared to the pure methanol activity set as a nonreducing control (0% reduction).Figure 5
Ferric-reducing activity assay. The % of reduced ferric ions byRubus caesius extracts is compared to the untreated control (0% reduction).We showed that the tested extracts (RC1–RC3) and subfractions (RC4–RC6) decreased the viability of cells, acting cytotoxically on tumour cells, and simultaneously expressed strong reductive activity. Our results are in agreement with studies by Durgo et al. [25], showing that red raspberry leaf extract expresses cytotoxic and antioxidative effects in the human colon adenocarcinoma (SW480) cell line. This activity was assigned mainly to polyphenolic compounds present in the plant material. Our results also confirmed results of Dai and coauthors, who revealed that the extracts from blackberry significantly limited HT29 human colon tumour cells growth, and the effect was dependent on the concentration applied. This effect was closely connected with the high content of anthocyanins [22]. Moreover, it was shown that acetone extract ofR. fairholmianus roots influenced human colon tumour cell morphology and reduced their viability via limitation of the intracellular ATP pool and changes in cells’ metabolic activity. As a consequence, depleted ATP quantity decreased the tumour cell proliferation rate and stimulated their death, mainly in the apoptotic pathway [28]. Furthermore, extracts from lyophilized fruits ofR. occidentalis may modulate host immune system processes by impacting on the function and viability of activated human CD4+ and CD8+ T lymphocytes [13]. It may indirectly influence tumour cell development and further metastasis. Analyses in this immunological direction have also been expanded with the use ofR. coreanus extracts loaded in gelatin nanoparticles. They were used as transport vehicles for the plant extracts and resulted in the significant enhancement of T, B, and NK cells’ functionality in all areas of their immune activity [14]. Interesting results using cold water extracts of fresh fruits ofR. caesius were shown by Turker et al. [29], who found a 100% antitumour efficiency of these extracts on cancer cells. In another study, similar effect was also supported by its antioxidative action with a relatively low IC50 value of 5 μg/mL [19]. Similarly, the shoot extracts ofR. idaeus were found to be a source of sanguiin H-6 and ellagic acid, which exhibit antioxidative as well as cytotoxic activity [18]. Lee and coauthors additionally revealed that sanguiin H-6 induces morphological changes in tumour cells which are similar to apoptotic features. However, this compound does not affect the cancer cell cycle. In general, the molecular pathway of sanguiin H-6 activity is mediated by MAPK p38 and BID cleavage with the participation of caspase-8 [20]. Kim and coworkers have shown that the aqueous extract of the incompletely ripened fruit ofR. coreanum inhibits cell proliferation and stimulates apoptosis in HT29 cells and that this may be mediated by its ability to activate the caspase-3 pathway [30]. In other study, Bowen-Forbes and coworkers showed that fruit extracts obtained from someRubus species also exhibited great potential to inhibit colon, breast, lung, and gastric cancer cell growth. The authors speculate that the anticancer effect may partially depend on inhibitory action on cyclooxygenase-2 (COX-2) functionality. Moreover, due to high anthocyanin content, it may also strongly influence the oxidative condition in the tumour cell microenvironment [23].Besides the general activity ofRubus extracts on tumour cells, it was shown that the range of such action is based on the horticultural parameters of the plant material. Production factors, both genetic and environmental, determine the usefulness of plants as a material for specific destiny, for example, chemoprevention. Therefore, the degree of inhibition of human colon tumour cell proliferation depends not only on general active phytoconstituents presence, but also on their specific composition which is dependent on cultivar, production site, or stage of maturity [21].Generally, plant extracts have many biological activities directly aimed at cell morphology and proliferation or indirectly by possessing reductive feature which influence inflammatory state modulating immune system reactivity. In our studyR. caesius leaf extract revealed tumour cell growth limiting activity, on both the morphology and metabolism levels. Moreover, its antioxidative activity may be connected with colon origin tumour cells growth reduction.



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