Although it has important pharmacological bioactivities, betulinic acid is still obtained by extraction from wood heart and bark and by transformation synthetically. With these methods, less than 3% efficiency is obtained. Our endemic rose species, Rosa pisiformis (Christ.) D. Sosn., which is a new alternative source of betulinic acid and traditionally used for its medicinal properties, was collected from the province of Gümüşhane, its natural distribution, and organs of the plant such as root, stem, leaf and fruit were air diried and pulverizated. The drugs are extracted separately with three solvents (methanol, dichloromethane and hexane) with Box-Behnken optimized extraction method and the amounts of quercetin, rutin, catechin and betulinic, ursolic and oleanolic acids in roots, stems, leaves and fruits are determined with standard substances using HPLC-DAD techniques. According to the Box-Benchen method optimisation data was obtained as 65% ratio of solvent and 5 times maceration with 75 ml of solvents. By the way the highest amount of catechin was found in the leaf (DCM) as 15.61 µg/ml; stem was found to be rich in rutin (28.96 µg/ml) and quercetin (39.90 µg/ml). The amount of betulinic acid in stem (hexane) (11.84 µg/ml) and root (9.32 µg/ml) has been determined for the first time, and their cytotoxic activities were determined mostly in stem also leaves and roots followed this subsequently against prostate and lung carcinoma cells by ABTS-assay.
Article
Application of Box-Behnken design for optimization of betulinic acid, ursolic acid and phenolics extraction from endemic Rosa psiformis subsp. pisiformis in relation to its cytotoxic activities
https://doi.org/10.21203/rs.3.rs-4287805/v1
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Rosa psiformis
betulinic acid
HPLC
GC-MS
Box-Benchen
Betulinic acid is a triterpenoid known to be isolated from different organs of various plants, including Rosa sp. This metabolite inhibits the growth of human melanoma [1] cells and the replication of the AIDS virus [2]. In addition, betulinic acid derivatives trigger apoptosis in human melanoma cells [3–5]. Despite such important pharmacological potential, betulinic acid is obtained by extraction from the bark or extracts of Betula alba and Betula pendula or by synthetic processes using betulin (alcoholic triterpene) isolated from the bark of these plants as an intermediate. Therefore, new research is required to identify new natural resources that can produce large quantities of crops from easily regenerated parts (leaves, lateral branches, fruit) without affecting plant growth. The aim of this study; The aim of this study is to determine and compare betulinic acid, oleanolic acid and ursolic acid amounts and phenolic compounds (catechin, rutin, and quercetin) in the root, stem, leaves and fruits of endemic Rosa pisiformis (Christ.) D. Sosn. (red rose). For this purpose, terpenes and phenolic compounds were isolated with 3 different solvent systems (hexane, dichloromethane and methanol) extraction technique from the roots, stems, leaves and fruits of the plant collected from the province of Gümüşhane, which is a natural distribution area, and structure and structure using GC-MS and HPLC-DAD techniques. quantitative determinations were made. While the rose plant is known with its significant amount of vitamin C especially in its fruits and seeds, also with antioxidant and anti-inflammatory properties; the presence of triterpene acids, especially betulinic acid, which has anti-cancer effect was determined in different organs (root, stem, leaf) besides fruit.
Rosehip species, rosehip, ascorbic acid, phenolic compounds and healthy fatty acids they contain have antioxidant properties and are also used in the treatment of skin disorders, hepatotoxicity, kidney diseases, diarrhea, inflammatory, arthritis, diabetes, hyperlipidemia, obesity and cancer diseases [6]. Betulinic acid, which has been determined to be present in various Rosa species [7], especially in Rosa canina [8], has attracted attention in recent years with its various biological and medicinal properties such as anti-bacterial, anti-malarial, anti-inflammatory, antihelmintic, antinociceptive, anti-HSV-I and anticancer effects [9]. Birch (Betula spp., Betulaceae) is being the main source of betulinic acid, a natural compound very common in the plant kingdom, but also found in Ziziphus spp. (Rhamnaceae), Syzygium spp. (Myrtaceae), Diospyros spp. (Ebenaceae) and Paeonia spp. (Paeoniaceae) species [10]. In phytochemical studies [11], conducted on 11 rose species distributed in the Eastern, Southern and Western regions of Turkey, total phenolic contents, vitamin C and antioxidant capacities of Rosa pisiformis, Rosa canina, Rosa villosa and Rosa dumalis subsp. antalyensis fruits were reported and antibacterial and antioxidant activities were determined. In addition, while it was determined that Rosa pisiformis fruits had the highest α-tocopherol (17.60 mg/g) value and the fructose ratio was 17.20 mg/g in fruits [12], there is no literature data on betulinic acid, ursolic acid and phenolics amounts. In this study, we aimed to determine the amount of betulinic acid ursolic acid and phenolics in different organs such as roots, stems, leaves, and fruits of our endemic species by Box-Benchen Method in relation to its cytotoxic activities.
Materials.
Plant. Materials (root, stem, leaf and fruit) of Rosa psiformis (Christ.) D. Sosn., collected from Gümüşhane (Akçakale, Gümüşhane-Turkey; 40°26'15.9”N, 39°30'46.8”E) and dried at room temperature (25°C) for 2 weeks. Dr. Volkan Eroğlu from Herbarium Center of Ege University undertook the formal identification of the plant material. Voucher specimen is kept in Ege University Herbarium Center with code number of 44010. The plant materials were ground after drying. It was stored at room temperature (25°C) until the time of extraction.
Chemicals and standards. Ethanol (HPLC grade), dichloromethane and hexane and standards of betulinic acid, oleanolic acid and ursolic acid and catechin, rutin and quercetin phenolics were obtained from (Sigma-Aldrich, Germany).
Cell lines for anticancer activity. Human prostate cancer cell line PC3 (PC-3) and A549 human lung carcinoma epithelia cells purchased from Atcc were treated. DMEM High Glucose (Capricorn CP 40-1309), %10 Fetal Bovine Serum (FBS, A0500-3010, Cegrogen Biotech, Germany), %0,5 Gentamisin 10 mg/mL (A2712 Merck, Germany), Sodyum pirüvat 100mM (L0473 Merck, Germany). Negative Control: %1 Dimethylsulfoxide (DMSO) contain nutrient medium. Blank: Culture prepared with cell culture medium without test substance.
Extraction and Box-Behnken method. The extraction process was carried out by maceration of 1 gr drug material in a 300 ml percolator one, three and five times. Box-Behnken (45) factorial trial design was used for each solvent and drug to optimize the extraction process (Ferreira et al., 2007). Drog/solvent ratio (65%, 80%, 95%), solvent (methanol, dichloromethane, hexane) and (50, 75, 100) solvent contents rating will be evaluated for root, stem, leaf and fruit (Table 1). Each factor will create 15 trial designs, low, medium, and high (− 1, 1, and 0) in triplicate. Coded value = Actual value − 0.5 × (high value + low value) 0.5 × (high value – low value)
Table 1. Extraction variables selected for Box-Bohnken Design (BBD) optimization.
All the meceration solvents are brought together and homogenized, filtered and brought to dryness in a rotary evaporator, and the percent yield is calculated, and then the planned trials for the amount of oleonolic, ursolic and betulinic acids are carried out. Maceration trials will be performed in triplicate and the values will be evaluated by giving the ± standard error of the mean. Data will be statistically evaluated at p < 0.05 using the t-test.
HPLC analysis of compounds. Tri Terpenes: HPLC Method was carried out by: Device: Agilent 1260 Infinity II, Column: Agilent Eclipse XDB- C18 column 5 µm 4,6*150 mm, Mobile Phase: %50 Asetonitrile: %50 methanol. Wavelength: 210 nm, Oven Temperature: 25˚C, Flow Rate: 0.5 mL/min, Injection Volume: 20 µL. Analysis were conducted by Ege MATAL (Ege University Central Research Test and Analysis Laboratory Application and Research Center).
Phenolics: was detected by HPLC Method; Device: Agilent 1260, Infinity II, Column: Agilent Eclipse XDB- C18 column 5 µm 4,6*150 mm, Mobile Phase: water containing 0.2% phosphoric acid 80%: Acetonitrile 20%, Wave Length: 210 nm, Oven Temperature: 25°C, Flow Rate: 1 mL/min, Injection Volume: 20 µL. Analysis were conducted by Ege MATAL (Ege University Central Research Test and Analysis Laboratory Application and Research Center).
Anticancer activity study. Cytotoxicity test were performed with A549, PC3 cells. Cytotoxic effect of the optimised plant material applied for 24 hours (in an environment of 5.0% CO2 and 95% humidity at 37°C) on cells planted at a concentration of 1x105 cells/mL on 96-well microplates was evaluated by reading their absorbance on a spectrophotometer (570 nm) with the MTT test (ISO 10993-5:2009). Cytotoxic activity identification studies were conducted by Ege MATAL (Ege University Central Research Test and Analysis Laboratory Application and Research Center). A statement to confirm that all experimental protocols were approved by a named institutional and/or licensing committee.
HPLC analysis of flavonoids and tri terpenes. Optimised dichlorometane (DCM) and ethanol (EtOH) extracts of root, stem, fruits and hexane extracts of fruit and leaf contain catechin, rutin and quercetin fenolics but only in stem and roots of R. pisiformis subsp. pisiformis rutin was not identified (Fig. 1). Betulinic and ursolic acids were the terpenes found in stems of R. pisiformis subsp. pisiformis (Table 2). Oleonolic acid was not identified in all the plant organs.
|
Solvent extracts |
Catechin (µg/mg) |
Rutin (µg/mg) |
Quercetin (µg/mg) |
Betulinic acid (µg/mg) |
Ursolic acid (µg/mg) |
|---|---|---|---|---|---|
|
DCM Fruit |
3,65 |
0,93 |
23,97 |
ni |
ni |
|
DCM Leaf |
15,61 |
12,80 |
20,70 |
ni |
ni |
|
DCM Stem |
3,56 |
28,96 |
39,90 |
ni |
ni |
|
DCM Root |
13,87 |
18,99 |
14,24 |
ni |
ni |
|
ETOH Fruit |
1,30 |
1,09 |
9,71 |
ni |
ni |
|
ETOH Leaf |
6,46 |
19,50 |
7,41 |
ni |
ni |
|
ETOH Stem |
7,13 |
10,05 |
11,51 |
ni |
ni |
|
ETOH Root |
1,57 |
0,30 |
3,45 |
ni |
ni |
|
Hexane Fruit |
0,94 |
0,66 |
2,71 |
ni |
59,10 |
|
Hexane Leaf |
1,45 |
0,18 |
2,95 |
ni |
171,50 |
|
Hexane Stem |
0,07 |
ni |
2,94 |
11,84 |
206,40 |
|
Hexane Root |
9,43 |
ni |
2,32 |
9,72 |
45,40 |
| DCM: dichloromethane, EtOH: ethanol | |||||
Cytotoxicity results
The presence of cytotoxic activities was not determined only in the DCM stem rich in catechin (3,56 µg/mg), rutin (28,96 µg/mg), and quercetin (39,90 µg/mg) of R. pisiformis subsp. pisiformis. Hexane extracts of root rich in catechin (9,43 µg/mg), quercetin (2,32 µg/mg), Betulinic acid (9,72 µg/mg), ursolic acid (45,90 µg/mg) and stem rich in catechin (0,07 µg/mg), quercetin (2,94 µg/mg), betulinic acid (11,84 µg/mg), ursolic acid (206,40 µg/mg) and also DCM leaf rich in catechin (15,61 µg/mg), rutin (12,80 µg/mg), quercetin (20,70 µg/mg) was cytotoxic according to the absorbances in the spectrophotometer (570 nm) with the MTT test (Graphic 1).
Table 3. The Box-Behnken response surface design and corresponding response values.
Box-Benchen experimental design. Box-Benchen experimental design. The content (mg) (Y1). The experiment running was showed in a raw with its responses [total content (Y1) and fenolic, terpenoid and flavonoid content (Y2)]. The reliability of the model fit is evaluated through the coefficient of variance (CV%), coefficient of determination (R2) and adjusted coefficient of determination (R2a). The modal capacity was determined by these tests to give the maximum mg and phenolic and terpenoid content.
The optimum total content
was found as 51,26 mg/g in fruit (ethanol), 40,93 mg/g in stem (dichloromethane), 44,28 mg/g in fruit (hexane). The results obtained from 5 days maceration by 65% ratio and 75 ml volume of solvent (Table 3).
For each response, the sequential model sum of squares and lack of fit tests were performed for quadratic, cubic, 2-factor interaction and linear models. While B2 had a directly proportional effect, C2 had an inversely proportional effect and was highly significant. A study was conducted to optimize the extraction process of phenolic and terpenoid compounds from the aerial parts of the plant, Rosa psiformis. The experiment involved four models (quadratic, cubic, 2-factor interaction, and linear) to determine the maximum yield and phenolic and terpenoid content. The effects of the factors and their interaction on each response were evaluated using analysis of variance. The results showed that the maceration time had the highest effect on increasing the total content, while the solvent/part of plant had a significant effect on the total phenolic content obtained. The model was validated through model accuracy and multiple response optimization. The maximum total content was found with 75 ml of 65% solvent at 5 days while the maximum phenolic content was found as 206,40 µg/mg ursolic acid, and 72,42 µg/mg phenolics (dichloromethane) and 11,84 µg/mg betulinic acid (hexane) in stem. The actual results were in close agreement with the predicted values, confirming the adequacy of the response model (Table 4, 5).
The three-dimensional (3D) plots were showed the effect of the interaction between total content and maceration time and solvent volume and the percentage of solvent in Fig. 2; phenolic and terpenoid content in Fig. 3. Phenolic and terpenoid content increased as a function of maceration time and solvent volume and the percentage of solvent resulted in an decrease also.
Table 4. Quadratic model fıtting for the total content *stastistically signifıcant: p<0.05.
Table 5. P-Values and estimated regression Coeffıcients of the two studied responses. *Statistically signifıcant: p<0.05.
In the world population approaching two billion, the fruits of rose plants (Rosa sp) contain polyphenols (flavonoids, proanthocyanidins, catechin), triterpene acids, essential fatty acids, galactolipid, folate, vitamins A, C and E, minerals (Ca, Mg, K, S, Si, Se, Mn and Fe) is a rich functional food [13]. The amount of ascorbic acid, which is good for Alzheimer's and cancer diseases, is 1200 mg/l for R. rugosa and 600 mg/l for R. canina fruits [14]. The main bioactive compounds isolated from this genus are flavonoids (eg kaempferol, quercetin, apigenin), triterpenoids (eg ursolic acid, tormentic acid, euscaphic acid, betulinic acid), and phytosterols (eg β-sitosterol) [15–22]. Triterpenes in rose fruits were determined as oleonolic acid, betulinic acid and ursolic acid [23].
R. canina L. fruit powders showed an immunomodulatory effect on human monocytic Mono Mac 6 cells. Triterpenic acid mixture (oleanolic, ursolic and betulinic acid) inhibited the release of IL-6 and TNF-α [24]. Botanical pentacyclic triterpenes have antioxidant, anti-tumor, anti-microbial and anti-inflammatory effects [25]. The mechanism of these bioactivities is believed to be due to the regulation of the immune system. As an example, treatment with a combination of compounds from natural products gives better responses than treatment with synthetic drugs alone, as examples of the combination of escin isolated from the seeds of Aesculus hippocastanum L. with a corticosterone, tumor necrosis factor alpha (TNF-α) (24.43%), interleukin 1-beta (Il). It has been found to reduce the synthesis of inflammatory markers such as -1β) (46.9%) and nitrite oxide (NO) (31.8%) [26]. In another study, it was proved that a mixture of oleonolic, ursolic and betulinic acid obtained from Rosa canina L. has a more effective immunomodulatory effect than a single compound [23]. It has been proven that the standard powder prepared from rosehip seeds and peels improves cell aging and skin wrinkles by accumulating in the membrane of erythrocyte cells [27].
Ursolic, oleanolic and betulinic acids are biologically active compounds in rosehip as immune system modulators [28, 29, 9]. Among the triterpenic acids, betulinic acid 36–772 mg/kg, oleanolic acid 66-1723 mg/kg and ursolic acid 37-2531 mg/kg were determined by negative ionization model. The amount of triterpenic acids varies between genotype, species and sections; It reaches the highest triterpene value (4600 mg/kg) in R. spinosissima. R. pulverulenta, which contains the lowest carotenoids, tocopherols and flavonoids, has a moderate triterpene acid content of betulinic acid 213 mg/kg, oleanolic acid 1045 mg/kg and ursolic acid 452 mg/kg [30]. Our results showed 11,84 µg/mg betulinic acid in stem of R. pisiformis subsp. pisiformis; 9,34 µg/mg in roots; 206,40 µg/mg ursolic acid in stem, 171,50 µg/mg in leaves, 59,10 µg/mg in fruit, 45,40 µg/mg in roots.
Some medically important secondary metabolites with antibacterial, anticancer, astringent, depurative and anti-inflammatory activities: polysaccharides, flavonoids, steroids, tannins, laevigatins (E, F, G), triterpenoids, 11α-hydroxytormentic acid, 2α-methoxyursolic acid, 6-methoxy-β-glucopyranosyl ester, tormentic acid and 5α-diol 3-O-β-d-glucopyranosides were determined [31]. In addition to flavonoids, triterpenoids eucaphic acid, nigaikigoside, betulinic acid, kajiikigoside, rubusid, tomentic acid and rosamutin were obtained from Rosa laevigata roots [32]. The presence of ursan-type triterpenes from Rosa cymosa Tratt used in Chinese herbal medicine roots was determined qualitatively and quantitatively by HPLC and TLC [33]. Two new triterpenes 19-oxo-18,19-seco-ursan-type lactone saponin and oleanin-type triterpenoid with anti-inflammatory effect were obtained from ethylacetate extracts of Rosa laevigata leaves [34]. Rosa rugosa var. plena and R. woodsii leaves were found to be rich in sesquiterpenes [35]. It has been reported that flavonoids, carotenoids and fatty acids in Rosa canina pseudo fruits have anti-inflammatory activity [36]. In Rosa pseudofructus cum/sine fructibus and Rosa canina L. fruits, triterpenoid acids ursolic, oleanolic and betulinic acids were detected in hexane and dichloromethane extracts and inhibited cyclooxygenases [9]. R. pisiformis subsp. pisiformis fruits were found to be rich ursolic asit (59,1 µg/mg) only and catechin (3,65 µg/mg), rutin (1,09 µg/mg), quercetin (23,97 µg/mg). Betulinic acid was not determined in fruits of R. pisiformis subsp. pisiformis. Cytotoxic activity was found only in leaf and root and stem of our endemic species R. pisiformis subsp. pisiformis.
The species of rose, rose hips, are wild plants mostly originating in Asia, less often in Europe, North America, and Northwest Africa, that have medicinal compounds traditionally used in the treatment of various diseases. While it is widely distributed in Turkey with 27 Rosa species, it is represented by two endemic species. Of these, Rosa pisiformis subsp. pisiformis (Christ.) D. Sosn. is endemic to Northeast and East Anatolia and in our provinces of Kars, Ağrı, Erzincan, Erzurum, Gümüşhane, Bayburt; Rosa dumalis BECHST subsp. boissieri (CREPIN) Ö. There is NILSSON. var. antalyensis (MANDEN.) Ö. NILSSON, on the other hand, shows a natural distribution in South West Anatolia.
Endemic Rosa pisiformis (Christ.) D. Sosn in its natural distribution; the evil eye rose (around Istanbul, in Van), rosehip (in the town of Güzelsu, Gürpınar district of Van), Şilan (in the Malazgirt district of Muş) [37, 38] is a medicinal perennial shrub belonging to the Rosaceae family. It originates in different parts of Europe, Asia and the America’s and Northwest Iran. The leaves and fruits of the plant contain high levels of vitamins (A, E, C), trace elements (Fe, Cu, Zn, Mn, Cr, Se) and minerals (Ca, K, Mg, Na). It is stated that it has protective effects on heart tissue antioxidant enzymes (GSH-Px, SOD, CAT) during stress [39]. Rosa pisiformis subsp. pisiformis (Christ.) D.Sosn leaves and fruits distributed in Van province have high levels of vitamins (A, E, C), trace elements (Fe, Cu, Zn, Mn, Cr, Se) and minerals (Ca, K, Mg, Na), however, it has been reported to have protective effects on cardiac tissue antioxidant enzymes (GSH-Px, SOD, CAT) during oxidative stress induced by isoproterenol [39]. Betulinic acid, an immune system modulator, has been proven to have anticancer effects on Rosa canina L. [25]. The fact that the seeds of Rosa canina and R. pisiformis contain higher amounts of fatty acids than their fruits [12] is important because fatty acids have the property of controlling the growth of pathogens and cancer cells [40]. In addition, another important feature of Rosa pisiformis seeds and fruits is that their vitamin E value is higher than Rosa canina. While the amounts of δ and α-tocopherol were (17.60 µg/g) in R. pisiformis; R. canina (7.15 µg/g) is also very low [12]. The fact that Rosa species has been investigated in terms of vitamin C and polyphenols and antioxidant activity directed us to investigate betulinic acid, one of the triterpenic acids, which has very important pharmacological effects such as anticarcinogens, qualitatively and quantitatively in endemic Rosa pisiformis subsp. pisiformis (Christ.) D. Sosn's different organs such as root, stem, leaf and fruit. According to the Box-Benchen method optimisation data was obtained as 65% ratio of solvent and 5 times maceration with 75 ml of solvents. By the way the highest amount of catechin was found in the leaf (DCM) as 15.61 µg/mg; stem was found to be rich in rutin (28.96 µg/mg) and quercetin (39.90 µg/mg). The amount of betulinic acid in (Hexane) stem (11.84 µg/mg) and root (9.72 µg/mg) has been determined for the first time, and their cytotoxic activities were determined mostly in (DCM) leaf rich in phenolics as catechine (15,61 µg/mg), rutin (12,80 µg/mg), quercetin (20,70 µg/mg) also in root (Hexane) and stem (Hexane) followed this subsequently against prostate and lung carcinoma cells by ABTS-assay.
Permission We have permission to collect Rosa psiformis (Christ.) D. Sosn. from General Directorate of Nature Conservation and National Parks in Turkey.
Data availability statement: The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.
Declarations Competing interests / COI statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported herein.
Funding: This research wassupported by Scientific Research Projects Council of Ege University with the Project number of FGA-2021-22385.
Author contributions
Hatice Demiray: concept, experimental design, finding grant, discussion, correction, and proofread. Mesut Emir Dibek: isolation, synthesis, structure elucidation, and drafting manuscript and discussion, anti-proliferative activity. Onur Özel: structure discussion, proofread.
Acknowledgements: Authorsare grateful to Scientific Research Projects Council of Ege University.
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Graphic 1 is available in the Supplementary Files section.
No competing interests reported.
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Graphical abstract
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Graphic 1. Cell viability results. Control, DCM Leaf, Terpene Root, Terpene Stem, DCM Stem.
