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Bioactivities of Alchemilla mollis, Alchemilla persica and Their Active Constituents

Abstract

The aim of this study was to determine antimicrobial activities of Alchemilla mollis, Alchemilla persica as well as ellagic acid and miquelianin against Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Candida albicans by using microbroth dilution method and anti-inflammatory activity by using human red blood cell (HRBC) membrane stabilization method. Microbroth dilution method was used to determine the antimicrobial activities. Extracts possessed activity having MIC values of 2.5-5-10mg/ mL, compounds possessed activity having MIC values of 1.25-2.5-4-5mg/mL. A.mollis aerial parts displayed the highest anti-inflammatory activity (IC50=1.22±0.07mg/mL). Ellagic acid and miquelianin were also determined as anti-inflammatory agents with 0.57±0.01mg/mL and 1.23±0.02mg/mL IC50 values, respectively. Total phenolic content and tannin content of the A.mollis and A.persica were determined as 357.00±75.80mg, 282.50±28.70mg PGE/g plant material and 18.02%, 18.63% respectively according to the method described by European Pharmacopoeia. Ellagic acid, miquelianin and catechin were analyzed by HPLC. The highest catechin content was detected in A.persica roots (6.69±0.05g/100g plant material). A.mollis aerial parts contain higher miquelianin (0.39±0.02g/plant material) and ellagic acid (1.56±0.01g/ plant material) than A.persica.

Keywords:
Alchemilla mollis; Alchemilla persica; Anti-inflammatory activity; Antimicrobial activity; Miquelianin; Ellagic acid; Biological activities of Alchemilla species

INTRODUCTION

The genus Alchemilla L. (Rosaceae) is distributed especially in Eurasia as well as in East Africa, North America, and Australia. In Europe and Mediterranean region, 541 species of this genus are recorded and 74 of them grow naturally in North and East Anatolia (Davis, 1982Davis PH. Flora of Turkey and the East Aegean Islands Vol 4. Edinburgh: Edinburgh University Press; 1982.; Hayırlıoğlu, Beyazoğlu, 1997Hayırlıoğlu S, Beyazoğlu O. Five new Alchemilla (Rosaceae) records for the flora of Turkey. Turk J Bot. 1997;21(5):309-312.; Özhatay, Kültür, 2006; Özhatay, Kültür, Gurdal, 2011; Aymerich, Sae, 2015Aymerich P, Sae L. Alchemilla cadinensis (Rosaceae), a new species from the Pyrenees (SW Europe). Wildenowia. 2015;45(3):435-442.).

Alchemilla vulgaris L. known as Lady’s mantle, Bear’s foot, Lion’s foot in Europe is the most studied species of Alchemilla genus. This plant is used in folk medicine internally for menopausal complaints, gynaecological disorders such as dysmenorrhea and menorrhagia, menstrual pain, gastrointestinal disorders, inflammation, weight loss, and externally as a gargle for mouth and throat inflammation as well as for wounds and skin disorders such as eczema and rashes (Said et al., 2011Said O, Saad B, Fulder S, Khalil K, Kassis E. Weight loss in animals and humans treated with “Weighlevel” a combination of four medicinal plants used in traditional Arabic and Islamic medicine. Evid Based Compl Alt Med. 2011;2011:1-6.; Ozbek et al., 2017Ozbek H, Bahadir-Acikara O, Keskin I, Kırmızı NI, Ozbilgin S, Ergene Oz B, et al. Evaluation of hepatoprotective and antidiabetic activity of Alchemilla mollis. Biomed Pharmacother. 2017;86:172-176.). European Pharmacopoeia described A. vulgaris aerial parts and Commission E approved usage of this plant for treatment of non-specific diarrhea (Blumenthal et al., 1998Blumenthal M, Busse WR, Klein J, Rister R, Hall T, Riggins C, et al. The Complete German Commission E Monographs. Therapeutic Guide to Herbal Medicines, 1st edition, Austin, TX, The American Botanical Council, 1998.). On the basis of clinical studies and long-term usage, A. vulgaris is suggested against non-specific diarrhea, gastrointestinal disorders and dysmenorrhea by ESCOP Monographs (Mills, Hutchins, 2013Mills MS, Hutchins R. ESCOPMonographs, Alchemillaherba- Alchemilla/Lady’s Mantle. European Scientefic Cooperative on Phytotherapy (ESCOP) Monographs, Online series. United Kingdom: ESCOP Notaries House; 2013.).

In Turkish folk medicine, Alchemilla species are used for wounds, skin disorders, gynaecological and menstrual complaints, diarrhea, liver inflammation, dyspnea, diabetes, asthma, bronchitis, kidney diseases, intestinal-gastric disorders and have sedative, tonic, diuretic, antitussive, anti-inflammatory properties (Akbulut, Bayramoğlu, 2013Akbulut S, Bayramoğlu MM. The trade and use of some medical and aromatic herbs in Turkey. EthnoMed. 2013;7(2):67-77.; Akbulut, Bayramoğlu, 2014Akbulut S, Bayramoğlu MM. Reflections of socio-economic and demographic structure of urban and rural on the use of medicinal and aromatic plants: the sample of Trabzon Province. EthnoMed . 2014;8(1):89-100.; Kalankan, Özkan, Akbulut, 2015Kalankan G, Ozkan ZC, Akbulut S. Medicinal and aromatic wild plants and traditional usage of them in Mount Ida (Balıkesir/Turkey). J Appl Biol Sci. 2015;9(3):25-33.; Küpeli Akkol et al., 2015Küpeli Akkol E, Demirel MA, Bahadır Acıkara O, Süntar I, Ergene B, Ilhan M, et al. Phytochemical analyses and effects of Alchemilla mollis (Buser) Rothm. And Alchemilla persica Rothm. In rat endometriosis model. Arch Gynecol Obstet. 2015;292(3):619-628.; Ozbek et al., 2017Ozbek H, Bahadir-Acikara O, Keskin I, Kırmızı NI, Ozbilgin S, Ergene Oz B, et al. Evaluation of hepatoprotective and antidiabetic activity of Alchemilla mollis. Biomed Pharmacother. 2017;86:172-176.).

Previous studies indicated that Alchemilla species have hepatoprotective (Ozbek et al., 2017Ozbek H, Bahadir-Acikara O, Keskin I, Kırmızı NI, Ozbilgin S, Ergene Oz B, et al. Evaluation of hepatoprotective and antidiabetic activity of Alchemilla mollis. Biomed Pharmacother. 2017;86:172-176.), angioprotective (Mills, Hutchins, 2013Mills MS, Hutchins R. ESCOPMonographs, Alchemillaherba- Alchemilla/Lady’s Mantle. European Scientefic Cooperative on Phytotherapy (ESCOP) Monographs, Online series. United Kingdom: ESCOP Notaries House; 2013.), vasorelaxant (Takır et al., 2015Takır S, Altun IH, Sezgi B, Süzgeç-Selçuk S, Mat A, Uydes- Doğan BS. Vasorelaxant and blood pressure lowering effects of Alchemilla vulgaris: A comparative study of methanol and aqueous extracts. Pharmacogn Mag. 2015;11(41):163-169.), antitumor (Mazzio, Soliman, 2009Mazzio EA, Soliman FK. In vitro screening for the tumoricidal properties of international medicinal herbs. Phytother Res. 2009;23(3):385-398.), cytotoxic (Türk et al., 2011Türk M, Kaya B, Menemen Y, Oguztuzun S. Apoptotic and necrotic effects of plant extracts belonging to genus Alchemilla L. species on HeLa cells in vitro. J Med Plants Res. 2011;5(18):4566-4571.), antioxidant (Ergene et al., 2010Ergene B, Bahadır Acıkara O, Bakar F, Saltan G, Nebioğlu S. Antioxidant activity and phytochemical analysis of Alchemilla persica Rothm. J Fac Pharm. 2010;39(2):145-154.; Mills, Hutchins, 2013Mills MS, Hutchins R. ESCOPMonographs, Alchemillaherba- Alchemilla/Lady’s Mantle. European Scientefic Cooperative on Phytotherapy (ESCOP) Monographs, Online series. United Kingdom: ESCOP Notaries House; 2013.), wound healing (Ergene Öz et al., 2016Ergene Öz B, İlhan M, Özbilgin S, Küpeli Akkol E, Bahadır Acıkara Ö, Saltan G, et al. Effects of Alchemilla mollis and Alchemilla persica on the wound healing process. Bangl J Pharmacol. 2016;11(3):577-584.), antimicrobial (Krivokuca et al., 2015Krivokuca M, Niketic M, Milenkovic M, Golic N, Masia C, Scaltrito MM, et al. Anti-Helicobacter pylori activity of four Alchemilla species (Rosaceae). Nat Prod Commun . 2015;10(8):1369-1371.) and antiviral properties (Ozbek et al., 2017Ozbek H, Bahadir-Acikara O, Keskin I, Kırmızı NI, Ozbilgin S, Ergene Oz B, et al. Evaluation of hepatoprotective and antidiabetic activity of Alchemilla mollis. Biomed Pharmacother. 2017;86:172-176.).

Alchemilla species are rich sources for phytochemicals such as tannins, flavonoids, proanthocyanidins, triterpenes and phenolic acids (Fraisse et al., 2000Fraisse D, Heitz A, Carnat A, Carnat AP, Lamaison JL. Quercetin 3-arabinopyranoside, a major flavonoid compound from Alchemilla xanthochlora. Fitoterapia. 2000;71(4): 463-464.; Olafsdottir, Omarsdottir, Jaroszewski, 2001Olafsdottir ES, Omarsdottir S, Jaroszewski JW. Constituents of three Icelandic Alchemilla species. BiochemSyst Ecol. 2001;29(9):959-962.; Trendafilova et al., 2011Trendafilova A, Todorova M, Nikolova M, Gavrilova A, Vitkova A. Flavonoid constituents and free radical scavenging activity of Alchemilla mollis. Nat Prod Commun . 2011;6(12):1851-1854.; Duckstein et al., 2013Duckstein SM, Lotter EM, Meyer U, Lindequist U, Stintzing FC. Phenolic constituents from Alchemilla vulgaris L. and Alchemilla mollis (Buser) Rothm. at different dates of harvest. J Biosci. 2013;68:529-540.).

In order to reveal wound healing activities of A.mollis and A.persica, which were established previously, due to inhibition of inflammation and prevention of microbial infections, we aimed to evaluate antimicrobial and anti- inflammatory activities in current study.

Chemical composition of Alchemilla mollis (Buser) Rothm. and Alchemilla persica Rothm. aerial parts and roots were investigated in this study in order to define responsible compounds for mentioned activities. Miquelianin (1) and ellagic acid (2) were isolated from the ethylacetate part of the methanol: water extracts of A.mollis aerial parts by using chromatographic techniques. Catechin, ellagic acid and miquelianin were analyzed by HPLC qualitatively and quantitatively. Antimicrobial activities of the A.mollis and A.persica as well as miquelianin and ellagic acid were evaluated against Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633,Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 10231. Anti-inflammatory activity of the extracts and isolated compounds were also tested by measuring their HRBC membrane stabilizing effects.

MATERIAL AND METHODS

Plant Material

A.mollis was collected from Sivas, Sarıyar in 2013, while A.persica was collected from Erzincan, Kop Passage in 2015. A.mollis was identified by Mehmet Tekin and deposited in Cumhuriyet University, Faculty of Science Herbarium (CUFH 1344). A.persica was identified by Hayri Duman and deposited in Ankara University Faculty of Pharmacy Herbarium (AEF 26371). Aerial parts and roots of the plants were dried at room temperature separately and then ground.

Extraction and Isolation

Dried and powdered aerial parts of A.mollis (995.96 g) were macerated with 1500 mL; roots of A.mollis (54.71 g), aerial parts of A.persica (51.71 g) and roots of A.persica (51.44 g) were macerated with 150 mL methanol:water (80:20) solvent system during 24 hours at room temperature separately, stirred in ultrasonic bath for 30 minutes and then filtered. Extracts were evaporated under vacuum at 35-45 °C and then lyophilized. Crude extract of A.mollis aerial parts (284.4801 g) was dissolved in water and subjected to liquid-liquid extraction by petroleum ether, chloroform and ethylacetate, to yield 5.4229 g; 15.0525 g; 64.7894 g of corresponding extracts, respectively.

Ethylacetate extract (21.4730 g) was applied to silica gel column (230-400 mesh, 0.040-0.063 mm, Merck ASTM) and eluted with ethylacetate:methanol:water (100:13.5:10) solvent system. Fractions were examined on TLC plates (Merck, 1.05554), with vanillin-sulfiric acid reagent and similar fractions were combined.

Fractions 14-16 obtained from silica gel column were applied to Sephadex LH-20 (25-100 μm, Sigma- Aldrich) column and eluted with methanol. Quercetin glucuronide, namely miquelianin(1), (16.1 mg) was obtained from Fractions 49-54, collected from Sephadex column as yellow precipitate. Fractions 34-46 obtained from silicagel column gave us ellagic acid (2) as a white precipitate (31.2 mg).

Identification

MS (Waters 2695 AlliaMicromass ZQ LC/MS, C-18 column), NMR1H (Varian Mercury 400, 400 MHz High Performance Digi NMR), 13C (Varian Mercury 400, 400 MHz High Performance Digi NMR) and 2D NMR (HMBC) (Varian Mercury 400, 400 MHz High Performance Digi NMR) were recorded to identify structure of the isolated compounds.

Antimicrobial Activity

Crude extracts and isolated compounds miquelianin and ellagic acid from aerial parts of A.mollis were dissolved in 20 % DMSO and were tested for their antimicrobial activities. Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 10231 were used for antibacterial and antifungal activity test respectively. Microbroth dilution method was used for determination of the minimum inhibitory concentrations (MIC). The cultures were obtained in Mueller Hinton Broth (Difco, Difco Laboratories, Detroit, MI, USA). Serial two-fold dilutions ranging from 10 to 0.078 mg/mL were prepared in medium. A set of wells containing only inoculated broth, 20 % DMSO, ampicillin, ofloxacin and fluconazole were used as control. After incubation for 18-24 h at 35±1°C for bacteria-48 h for fungi, the last well with no microbial growth was recorded to represent MIC value (mg/mL).

Anti-inflammatory Activity

The study protocol was approved by the ethics committees of the Faculty of Medicine of Ankara University, Ankara-Turkey (26.10.2015/16-695-15).

Preparation of Human Red Blood Cells (HRBC) Suspension: Fresh whole human blood was collected from healthy human volunteer who had not taken any anti-inflammatory or steroidal drug for 2 weeks prior the experiment and transferred to the centrifuge tubes. The tubes were centrifuged at 3000 rpm for 10 min. The packed cells were washed at least three times with equal volume of isosaline (0.85 %, pH7.2). The volume of the blood was measured and reconstituted as 10 % v/v suspension with isosaline.

Heat Induced Hemolysis: Membrane stabilizing activity of the extracts was assessed using heat-induced human erythrocyte hemolysis. A stock solution of each sample was prepared by dissolving 20 mg of extract/ acetylsalicylic acid with 1 ml of their solvents. The reaction mixture consisted of test sample (extracts) and 10 % HRBC suspension. Instead of test sample, only solvent was used as a control. Acetylsalicylic acid was used as a standard drug. All centrifuge tubes containing reaction mixture were incubated in water bath at 56 ºC for 30 min. Then, the incubation tubes were cooled under running tap water. The reaction mixture was centrifuged at 2500 rpm for 5 min. The absorbance of the supernatant was measured at 560 nm (Anosike, Obidoa, Ezeanyika, 2012Anosike CA, Obidoa O, Ezeanyika LUS. Membrane stabilization as a mechanism of the anti-inflammatory activity of methanol extract of garden egg (Solanum aethiopicum). DARU J Pharm Sci. 2012;20(1):76.). Then, the efficient concentrations of the samples were determined by diluting the solution and the half maximal inhibitory concentration (IC50) values were calculated.

Statistical analyses

All the experiments were performed at least in triplicates for all the test samples. SPSS 23.0 was used to examine the results. Results were expressed as mean and standard deviation (SD). One-way analysis of variance was performed by ANOVA procedures. Significant differences between means were determined by the Fisher’s least significant difference (LSD) test. P values less than 0.05 were reported as “statistically significant”.

Total Phenolic Content

Total phenol content of the aerial parts and roots of A.mollis and A.persica was carried out using the method described in European Pharmacopeia 6.0. Aerial parts and roots of A.mollis and A.persica were powdered. 0.5 g of each material was heated with 150 mL water at water-bath for 30 minutes, then diluted to 250 mL with water and filtered. First 50 mL of the solution was discarded. 5 mL of each extract was diluted to 25 mL with water. Mix 2 mL of each solution with 1 mL phosphomolybdotungstic reagent (Sigma-Aldrich) and 10 mL water, then diluted to 25 mL with 29 % sodium carbonate (Atabay) solution. After 30 minutes final mixture’s absorbance was measured at 760 nm as A1 against the water as a blank solution.

Total phenolic content of the extracts were expressed as gallic acid and pyrogallol equation. For pyrogallol equation, 50 mg pyrogallol was dissolved in water and diluted to 100 mL with water. 5 mL of the solution was diluted to 100 mL with the same solvent. 2 mL of this solution was mixed with 1 mL phosphomolybdotungstic reagent (Sigma-Aldrich) and 10 mL water, then diluated to 25 mL with 29 % sodium carbonate (Atabay) solution. After 30 minutes, absorbance was read at 760 nm as A3, against the water. Following equation was used for calculation of total phenolic content.

Total phenolic content = 62 . 5 × A 1 × m 2 A 3 × m 1

m1 = weight of test material (g),

m2 = weight of pirogallol (g)

For gallic acid equation, standard compound, gallic acid was weighed as 50 mg and dissolved in 10 mL of water. Different concentrations of standard compound were prepared (0.5, 0.1, 0.05, 0.025 mg/ mL). 2 mL of each concentration was mixed with 1 mL phosphomolybdotungstic reagent (Sigma-Aldrich) and 10 mL water, then diluted to 25 mL with 29 % sodium carbonate (Atabay) solution. After 30 minutes, final mixture’s absorbance was measured at 760 nm against the water as blank solution. Calibration curve was drawn and the equation was calculated. Linear correlation between concentration and absorbance was expressed as y = mx + n equation. Total phenolic content of the tested extracts was calculated by the following equation y=1.417x+0.067. y-absorbance, x-total phenolic content as gallic acid mg/g plant material equivalent.

Tannin Content

Tannin content of the aerial parts and roots of A.mollis and A.persica was carried out using the method described in European Pharmacopeia 6.0. Aerial parts and roots of A.mollis and A.persica were powdered. 0.5 g of each material was heated with 150 mL water at water-bath for 30 minutes, then diluted to 250 mL with water and filtered. First 50 mL of the solution was discarded. 5 mL of each extract was diluted to 25 mL with water. 2 mL of each solution was mixed with 1 mL phosphomolybdotungstic reagent (Sigma-Aldrich) and 10 mL water, then diluted to 25 mL with 29 % sodium carbonate (Atabay) solution. After 30 minutes, final mixture’s absorbance was measured at 760 nm as A1, against the water as blank solution. 10 mL of each extract was stirred with 0,10 g hide powder for 60 minutes and filtered. 5 mL of each extract was made up to 25 mL with water. 2 mL of each solution was mixed with 1 mL phosphomolybdotungstic reagent (Sigma-Aldrich) and 10 mL water, then diluted to 25 mL with 29 % sodium carbonate (Atabay) solution. After 30 minutes, final mixture’s absorbance was read at 760 nm as A2, against the water as blank. Tannin content of the extracts was calculated from following equation and expressed as pyrogallol equation.

Tannin content = 62 . 5 × A 1 - A 2 × m 2 A 3 × m 1

For determining tannin content as gallic acid, equivalent calibration curve of gallic acid was used and each extract was stirred with hide powder using the method described in European Pharmacopeia (2008European Pharmacopoeia 6.0 (Vol.2). Alchemillae Herba.01/2008: 1123. Germany: Directorate for the Quality of Medicines & HealthCare of the Council of Europe (EDQM), 2008.) and final mixture’s absorbance was read at 760 nm as phenolic compounds were not absorbed by hide powder against the water as blank. Tannin content of the extracts was calculated from the same equation by subtracting absorbance of phenolic compounds not absorbed by hide powder from absorbance of total phenolic content.

HPLC Analyses

HPLC analyses were carried out using Agilent LC 1260 chromatograph (Agilent Technologies, Darmstadt, Germany). The diode array detector (DAD) was set at wavelengths of 210 and 254 nm and peak areas were integrated automatically using Agilent Chem Station Software. ACE 5 C18 (250 mm; 4.6 mm; 5 µm) column was used for separation. The mobile phase was made up of acetonitrile (A) and 0.2 % phosphoric acid (B) using gradient elution: initial percentage A:B (8:92, v/v), then 0- 20 min linear change from A:B (8:92, v/v) to A:B (15:85, v/v), and 20-40 min linear change from A:B (15:85, v/v) to A:B (25:75, v/v) were used. This was followed by gradient elution from A:B (25:75, v/v) to A:B (50:50 v/v) in 40-50th min and from A:B (50:50 v/v) to A:B (100:0, v/v) in 50- 55th min. Isocratic flow A:B (100:0,v/v) was applied from 55th min to 60th min. The flow rate was 0.5 mL/min, column temperature was maintained at 25 ºC. The sample injection volume was 10 µL. Both aerial parts and roots of A. mollis and A. persica (0.5 g) were extracted with methanol:water (80:20) mixture in ultrasonic bath for 30 min. After filtration, all extracts were adjusted to 10 mL in volumetric flask. Each extract was filtered from 0.45 μm filters before injection to HPLC system. Catechin, gallic acid, chlorogenic acid and caffeic acid purchased from Sigma; ellagic acid and miquelianin obtained from A. mollis were used as standard compounds. To prepare calibration curves, peak area of each solution was plotted against the concentration. Tested compounds were prepared at six different concentration levels (0.01, 0.02, 0.05, 0.1, 0.2, 0.25 mg/mL). Triplicate injections of 10 µL of each standard solution were performed. The compounds were determined at 210 and 254 nm absorbances. LOD (Limit of detection) and LOQ (Limit of quantification) of each compound were determined as signal to noise ratio of 3 and 10 respectively and verified by six injections at 10 µL.

RESULTS AND DISCUSSION

The MIC values (mg/mL) of the methanol:water extracts of A. mollis and A. persica as well as miquelianin and ellagic acid were shown in Table I. Neither extracts nor compounds showed antibacterial activity against E.coli. Both of the aerial part extracts of the tested plants and also miquelianin didn’t show antibacterial activity against B. subtilis. Root extract of A. mollis, ellagic acid and miquelianin didn’t show antibacterial activity against E. faecalis. No antibacterial activity was observed against P. aeruginosa for ellagic acid and no antifungal activity was observed against C. albicans for root extract of A. mollis. Except these, extracts possessed activity having MIC values of 2.5-5-10 mg/mL, miquelianin and ellagic acid possesed activity having MIC values of 1.25-2.5-4-5 mg/mL against the tested microorganisms.

TABLE I
Minimum Inhibitory Concentration values (mg/mL) of Alchemilla species and isolated compounds against tested microorganisms

Anti-inflammatory activity of the samples was determined by measuring stabilization capacity against heat induced hemolysis of HRBC membrane. The extracts showed significant protection of the HRBC membrane against hemolysis induced by heat compared to acetylsalicylic acid used as a reference drug. A.mollis aerial part extract (IC50=1.22±0.07 mg/mL) exhibited the highest HRBC membrane stabilizing effect almost 4 times lower than acetylsalicylic acid (IC50=0.29 mg/mL) (Table II).

TABLE II
Human red blood cell membrane stabilizing effects of methanol:water extracts of Alchemilla species and isolated compounds.

Total phenol and tannin content of the A. mollis and A. persica was tested according to the method described in European Pharmacopoeia 6.0 in current study. Gallic acid and pyrogallol are both used as standards. Results of our analysis revealed that the total polyphenol content of A. persica and A. mollis varied from 244±72.7 to 331.7± 117.7 mg GAE/ g and tannin content varied from 8.3±4.42 to 12.95±3.00 g GAE/100 g plant material. As pyrogallol equivalent, total phenolic content varied from 237.8±25.9 to 357±75.8 mg PGE/ g and tannin content between 9.35± 0.29 and 18.63 ±0.29 g PGE/100 g plant material. The highest tannin and total phenolic content as pyrogallol equivalent were determined for both aerial parts of A.mollis and A. persica extracts (Table III and Table IV). Our research reveals that aerial parts of A.mollis exhibited the highest red blood cell membrane stabilization activity (IC50: 1.22±0.07 mg/mL) with a good protection against heat-induced lysis and followed by A.mollis root extract (IC50: 1.34±0.08 mg/mL). These results can be attributed to the higher phenolic and tannin content.

TABLE III
Total phenolic content and tannin content of Alchemilla mollis and Alchemilla persica as gallic acid equivalent

TABLE IV
Total phenolic content and tannin content of Alchemilla mollis and Alchemilla persica as pyrogallol equivalent

Total phenol contents of the A. mollis and A. persica were also determined and the highest content was observed in A. persica roots (331.69±117.27 mg GAE/ g plant material; 282.50±28.70 mg PGE/g plant material). Ellagic acid and miquelianin as well as catechin were also analysed by HPLC qualitatively and quantitatively (Table V) LOD and LOQ values of catechin, miquelianin and ellagic acid were also determined and are given in Table VI. Table V exhibits both roots of A. mollis and A. persica, which were found to contain catechin while aerial parts did not. The highest content of the catechin was determined in A. persica roots (6.69±0.05 g/100 g plant material). On the other hand, ellagic acid and miquelianin were only detected in aerial parts of both species. Ellagic acid and miquelianin were detected as major compounds in HPLC chromatogram of the A. mollis and A. persica aerial part extracts. Miquelianin percentages of the both species were higher than A. vulgaris. However, ellagic acid content of the A. persica was lower than A. vulgaris while A. mollis contained a higher amount. Both miquelianin and ellagic acid were isolated from ethylacetate part of the methanol-water extract of A. mollis aerial parts using different chromatographic techniques in the present study. Structures of the isolated compounds were determined according to the NMR1H and 13C NMR results and comparing with literature data (Li et al, 1999Li XC, Elsohly HN, Hufford CD, Clark AM. NMR assignments of ellagic acid derivatives. Magn Reson Chem. 1999;37(11):856-859.; Nugroho, Lee, 2014Nugroho A, Lee KT. Quantification of antidepressant miquelianin in mature and immature fruits of Korean Rubus species. Nat Prod Sci. 2014;20(4):258-261.; Vijayalakshmi, Madhira, 2014Vijayalakshmi A, Madhira G. Anti-psoriatic activity of flavonoids from Cassia tora leaves using the rat ultraviolet B ray photodermatitis model. Rev Bras Farmacogn. 2014;24(3):322-329.). Previous studies have revealed that A. mollis contains miquelianin, hyperoside, isoquercetin, cis- and trans-tiliroside, sinocrassoside D2, rhodiolgin and gossypetin-3-O-β-D-galactopyranosyl- 7-O-α-L-rhamnopyranoside (Trendafilova et al., 2011Trendafilova A, Todorova M, Nikolova M, Gavrilova A, Vitkova A. Flavonoid constituents and free radical scavenging activity of Alchemilla mollis. Nat Prod Commun . 2011;6(12):1851-1854.). In acetone/water extracts of A. mollis, pedunculagin, agrimoniin, sanguiin H-10, castalagin/vescalagin as monomeric and oligomeric ellagitannins as well as galloyl-bis-hexahydroxydiphenoyl (HHDP) hexose, gallic acid, chlorogenic acid, catechin, a procyanidin trimer, quercetin glucuronide (miquelianin), several other quercetin glycosides, kaempferol glucuronide and methylated quercetin glucuronide were determined by liquid chromatography-tandem mass spectrometry (LC- MS/MS) (Duckstein et al., 2013Duckstein SM, Lotter EM, Meyer U, Lindequist U, Stintzing FC. Phenolic constituents from Alchemilla vulgaris L. and Alchemilla mollis (Buser) Rothm. at different dates of harvest. J Biosci. 2013;68:529-540.). Sesquiterpenes and diterpenes as well as phenolic compounds including catechin, gallic acid, chlorogenic acid, gallic acid glycoside, gallic acid methoxy glycoside, pedunculatin isomers, galloyl-HHDP-hexoside isomers, epicatechin, quercetin-3-O-glucuronide, sanguiin isomers, casuarictin, agrimoniin, procyanidin B1, kaempferol- 3-O-rutinoside, methyl gallate, ellagic acid, digalloyl- galloylgalloside, aromadendrin glucoside derivative were determined in A. persica using GC-MS and HPLC-MS techniques (Afshar et al., 2015Afshar FH, Maggi F, Ferrari S, Peron G, Dall’Acqua S. Secondary Metabolites of Alchemilla persica Growing in Iran (East Azarbaijan). Nat Prod Commun. 2015;10(10):1705-1708.). Ellagic acid was isolated from A. mollis aerial parts for the first time in the current study. European Pharmacopoeia describes A. vulgaris dried leaves as “Alchemillae herba” an official pharmaceutical drug. According to the European Pharmacopoeia and ESCOP monographs, Alchemillae herba should contain minimum 6.0 per cent of tannins expressed as pyrogallol, approximately 2.2-2.5 % flavonoid glycosides and the miquelianin namely as main flavonoid (European Pharmacopoeia, 2008European Pharmacopoeia 6.0 (Vol.2). Alchemillae Herba.01/2008: 1123. Germany: Directorate for the Quality of Medicines & HealthCare of the Council of Europe (EDQM), 2008.; Mills, Hutchins, 2013Mills MS, Hutchins R. ESCOPMonographs, Alchemillaherba- Alchemilla/Lady’s Mantle. European Scientefic Cooperative on Phytotherapy (ESCOP) Monographs, Online series. United Kingdom: ESCOP Notaries House; 2013.). Additionally, according to Fraisse et al. (2000Fraisse D, Heitz A, Carnat A, Carnat AP, Lamaison JL. Quercetin 3-arabinopyranoside, a major flavonoid compound from Alchemilla xanthochlora. Fitoterapia. 2000;71(4): 463-464.), levels of the total flavonoids were detected as 2.22 %, miquelianin 1.18 %, tannins 16.0 % and ellagic acid 0.36 % for A. vulgaris.

TABLE V
Quantity of ellagic acid, miquelianin and catechin in plant materials

TABLE VI
Limit of Detection and Limit of Quantition values of ellagic acid, miquelianin and catechin

According to the literature survey, antibacterial activities of the A. mollis were tested against S. aureus, S. S. epidermidis, S. pyogenes, P. aeruginosa, K. pneumonia and E. coli previously. Water, ethanol and methanol extracts of the A. mollis displayed antibacterial activity against E. coli and P. aeruginosa in disc diffusion and well diffusion assays. Inhibition zones were determined as 9.33 ± 0.66 mm, 15.33 ± 0.66 mm and 9.33 ± 0.66 mm for water, ethanol and methanol extracts against E. coli and 22.67 ± 0.66 mm, 22.67 ± 1.33 mm for ethanol and methanol extracts against P. aeruginosa in disc diffusion test. Ethanolic and methanolic extracts of A. mollis antimicrobial activity were similar or greater than tested antibiotics; ovobiocin (23 mm), carbenicillin (22 mm), tetracycline (13 mm), bacitracin (11 mm), ampicillin (8 mm) and lincomycin (7 mm) in disc diffusion methods against P. aeruginosa. Same results were obtained in well diffusion method (Usta, Birinci Yıldırım, Uçar Türker, 2014Usta C, Birinci Yıldırım A, Uçar Türker A. Antibacterial and antitumour activities of some plants grown in Turkey. Biotechnol Biotech Eq. 2014;28(2):306-315.). Additionally, A. mollis was tested against S. aureus, E. coli, Salmonella thphimurium, Salmonella enteritidis and C.albicans aureus in disc diffusion and micro dilution assays. Ethyl acetate, methanol, butanol, 70% methanol and water extracts of the A. mollis displayed higher antibacterial activity against S. aureus and Sa.enteritidis. Inhibition zones were determined as 19.00 ± 00 mm, 18.66 ± 0.57 mm, 18.66 ± 1.52, 13.33 ± 1.15 and 19.00 ± 2.00 mm for ethyl acetate, methanol, butanol, 70% methanol and water extracts against S. aureus in disc diffusion test. Antimicrobial activity was similar in tested antibiotic; penicillin (19 mm) against S. aureus. Ethyl acetate, hexane, methanol, butanol, 70% methanol and water extracts showed moderate activity against E.coli and Sa.typhimurim. Inhibition zones were observed as 13.66 ± 1.05, 16.33 ± 3.05, 12.16 ± 1.04, 11.00 ± 00, 14.00 ± 2.64 and 9.33 ± 0.57 mm for ethyl acetate, methanol, butanol, 70% methanol and water extracts respectively and 21.00 ± 1.00 mm for penicillin against E.coli. Ethyl acetate, methanol and water extracts of A. mollis showed moderate antimicrobial activity against C.albicans with ranging MIC values from 125 to 500 µg/mL. (Şeker Karatoprak, İlgün, Koşar, 2017Şeker Karatoprak G, Ilgun S, Kosar M. Phenolic composition, anti-inflammatory, antioxidant, and antimicrobial activities of Alchemilla mollis (Buser) Rothm. Chem Biodiversity. 2017;14(9):1-12.). Our study results are not similar for antibacterial activity against E. coli. On the other hand, antimicrobial activity was observed against P. aeruginosa with 5-10 mg/mL MIC values. The highest activity was observed with A. persica aerial part extract (MIC 5 mg/mL). Miquelianin showed antimicrobial activity against Streptococcus mitis, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum with 0.25 mg/ mL MIC values (Shahzad et al., 2015Shahzad M, Millhouse E, Culshaw S, Edwards CA, Ramage G, Combet E. Selected dietary (poly)phenols inhibit periodontal pathogen growth and biofilm formation. Food Funct. 2015;6(3):719-729.). Ellagic acid was determined as one of the active constituents of Syzygium aromaticum for its antimicrobial activity against oral pathogens such as Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans, Actinomyces viscosus with 1.25 mg/mL MIC value (Cai, Wu, 1996Cai L, Wu CD. Compounds from Syzgium aromaticum posessesing growth inhibitory activitiy against oral pathogens. J Nat Prod. 1996;59(10):987-990.). It has been reported that ellagic acid exhibited antibacterial activity against S.aureus ATCC 9538, S.epidermidis NCTC 11047, Micrococcus luteus NCTC 9341, E.coli NCTC 8196, B.subtilis ATCC 6633 with 0.63 mg/mL, 0.31 mg/mL, 0.31 mg/mL, 2.5 mg/mL, 0.63 mg/mL and antifungal activity against C.albicans ATCC 10231 with 1.25 mg/ mL MIC values (Thiem, Goslinska, 2004Thiem B, Goslinska O. Antimicrobial activity of Rubus chamaemorus leaves. Fitoterapia. 2004;75(1):93-95.). Present study results revealed that similar results were obtained for ellagic acid. This compound displayed antibacterial activity against B. subtilis and S. aureus with 5 mg/mL, 1.25 mg/mL MIC values respectively and antifungal activity against C. albicans 2.5 mg/mL MIC value as shown in Table I. Martini et al. (2009Martini S, D’Addario C, Colacevich A, Focardi S, Borghini F, SantucciFigura N, et al. Antimicrobial activity against Helicobacter pylori strains and antioxidant properties of blackberry leaves (Rubusulmifolius) and isolated compounds. Int J Antimicrob Ag. 2009;34(1):50-59.) have reported that antimicrobial activity of ellagic acid on H.pylori G21 and H.pylori 10K was also observed at 0.002 mg/mL and 0.01 mg/mL concentrations, respectively. Ellagic acid inhibited growth of oral pathogens such as Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius, Actinomyces naeslundii, Actinomyces viscosus, Lactobacillus rhamnosus, Porphyromonas gingivalis and Bacteroides forsythus (Loo et al., 2010Loo WTY, Jin LJ, Cheung MNB, Chow LWC. Evaluation of Ellagic acid on the activities of oral bacteria with the use of adenosine triphosphate (ATP) bioluminescence assay. Afr J Biotechnol. 2010;9(25):3938-3943.). Antibacterial activity of ellagic acid against Staphylococcus epidermatis, Bacillus cereus, Klebsiella pneumonia and Salmonella typhi was also tested using plate-agar method. Activity of the compound was determined as more effective than gentamycin and streptomycin. Minimum effective dose of ellagic acid was determined as 0.15 mg/mL while gentamycin and streptomycin were 0.3 mg/mL and 2 mg/ mL respectively (Ghudhaib, Hanna, Jawad, 2010Ghudhaib KK, Hanna ER, Jawad AH. Effect of ellagic acid on some types of pathogenic bacteria. JNUS. 2010;13(2):79-85.).

According to current study results, A.mollis aerial parts contain tannin, miquelianin and ellagic acid in a proper percentage when compared to A.vulgaris as an official pharmaceutical drug. A. persica also contains a higher amount of tannin and miquelianin than A. vulgaris and a lower amount of ellagic acid. These results may explain the traditional usage of A. mollis in European traditional medicine for same purposes with A. vulgaris as a commercial drug “Herba Alchemillae” for its astringent, diuretic, antispasmodic, wound healing activities and against excessive menstruation (Trendafilova et al., 2011Trendafilova A, Todorova M, Nikolova M, Gavrilova A, Vitkova A. Flavonoid constituents and free radical scavenging activity of Alchemilla mollis. Nat Prod Commun . 2011;6(12):1851-1854.; Özbek et al., 2017).

In conclusion, antimicrobial activity was observed with aerial parts and roots of A. mollis and A. persica as well as miquelianin and ellagic acid as isolated compounds against tested microorganisms. All the plant extracts tested as well as ellagic acid and miquelianin stabilized the HRBC membrane against heat significantly. Miquelianin, ellagic acid and tannin content of the Alchemilla species could be responsible for their antimicrobial and anti-inflammatory activities. Additionally, tannin and miquelianin content of A. mollis was determined as high as A. vulgaris. These results may suggest the usage of A. mollis instead of A. vulgaris in countries where A. vulgaris does not grow naturally.

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Publication Dates

  • Publication in this collection
    14 Mar 2022
  • Date of issue
    2022

History

  • Received
    08 Jan 2018
  • Accepted
    10 June 2020
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br