Diuretic activity and toxicity of some Verbascum nigrum extracts and fractions

Abstract

Context. Verbascum nigrum L. (Scrophulariaceae) is a perennial plant used in folk medicine for the treatment of kidney diseases due to its presumable diuretic properties.

Objective: We investigated the diuretic activity and toxicity of extracts from different parts of V. nigrum and identified a group of compounds responsible for the biological effect.

Materials and methods: Five ethanol extracts from herb, roots, flowers, leaves and stems as well as five fractions of polar compounds isolated from herb of V. nigrum were orally administrated as a single dose of 50 mg/kg to rats. Urinary excretion and electrolyte content were measured at 3 and 6 h after the treatment. The acute toxicity of the V. nigrum extracts and fractions was evaluated in mice.

Results: All extracts, except the one prepared from the roots, showed a significant increase of the urine output within first 3 h after their administration. The extract from stems was the most active, inducing urine output of 14.6 ± 0.8 ml/kg BW versus 5.2 ± 1.4 ml/kg BW of the control. It also demonstrated saluretic activity with a natriuretic index 4.1 and a kaliuretic index 3.8. The diuretic activity was correlated with the flavonoid content in the plant organs. Flavonoid fractions demonstrated significant activity; the higher content of flavonoids (expressed as hesperidin) translated into more pronounced diuretic (35.9 ± 2.1 ml/kg BW) and saluretic effects (natriuretic index 4.5 and kaliuretic index 5.4).

Conclusion: The diuretic activity of traditionally used V. nigrum was validated experimentally. The pharmacological effect was attributed to flavonoids, which accumulated in aerial parts of the plant, mainly in stems.

• Diuresis
• electrolyte excretion
• flavonoids
• hesperidin
• mullein
• Scrophulariaceae

Introduction

Plants are considered as one of the main sources of biologically active chemicals. Despite the recent domination of synthetic chemistry in the field of discovering and producing new drugs, the potential of the plant extracts and their bioactive constituents to provide novel products for disease treatment and prevention remains enormous (Barnes et al., 2007). Diuretics are commonly used in therapies of hypertension, edema, renal disease and other problems associated with water retention (Ramelet, 2000; Williams et al., 2004). Herbal diuretics have a very wide range of indications and are especially preferred for therapy of chronic conditions. Advantages of using diuretics of herbal origin lie in their mild therapeutic effect, low toxicity, minimal side effects and affordability.

Some plants of the Scrophulariaceae family, growing wild in many parts of Eurasia, Africa, and North America, have been reported to possess diuretic effect (Shishkin & Bobrov, 1955). The Verbascum genus of the Scrophulariaceae family consists of about 360 species (Heywood, 1993). The most explored and approved for use in official medicine is Verbascum thapsus L., which has been proved to be effective in the treatment of some pulmonary problems. The pharmacological investigations of this plant revealed that the soothing effect on the mucus membranes could be attributed to the mucilaginous constituents, while the expectorant action seemed to be due to the V. thapsus saponins (Tyler, 1993). The antiviral, antibacterial and antifungal activities of the extract from the V. thapsus leaves were reported (McCutheon et al., 1992, 1994, 1995). Verbascum thapsus also demonstrated mild diuretic activity with soothing and anti-inflammatory effects on the urinary tract (Tyler, 1994). Herein we report results of our studies on a less explored species of the Verbascum genus, V. nigrum (black mullein). This plant is widely distributed in Eurasia and has a long history of traditional use in many folk medicine systems (Baytop, 1999; Leporatti & Ivancheva, 2003). In Russia, V. nigrum is popularly known as “Tsarsky skipetr” (Czar’s scepter). This plant has found many applications in Russian traditional folk medicine. Ethanol and aqueous extracts of V. nigrum are used for treating neurosis, epilepsy, acute respiratory diseases, asthma, diarrhea, gastralgia and dysentery (Budantsev & Lesovskii, 2001). Infusion of black mullein has also been used in folk medicine as a diuretic medication for the treatment of kidney diseases (Sokolov, 1990).

In pharmacological investigations, dry extract of the black mullein herb has been shown to possess anti-inflammatory, anticoagulant (Ustinova et al., 2009) and hypotensive (Kalinina et al., 2011) activities. The pharmacological effects of the extract have been attributed to various biologically active chemical constituents of the plant, predominantly flavonoids and iridoids. Danchul (2007) reported the following flavonoid composition for black mullein: hesperidin and quercetin, glucuronides of luteolin, apigenin, diosmin and quercetin. Iridoids harpagoside and aucubine were also found in the plant extracts (Danchul et al., 2006; Seifert et al., 1982; Vesper & Seifert, 1994).

Our study evaluates the diuretic activity and toxicity of extracts from herbs and different parts of V. nigrum in comparison with natural diuretic preparations used in Russian official medicine, i.e., extracts of Equisetum arvense L. (Equisetaceae) and Vaccinium vitis-idaea L. (Ericaceae) and the standard diuretic drug furosemide. In order to identify group(s) of compounds responsible for the diuretic effect, the herb extract from V. nigrum was subjected to fractionation using column chromatography and biological activity of the obtained fractions was evaluated.

Materials and methods

Hesperidin, quercetin and luteolin standards for chromatographic analysis were purchased from Sigma-Aldrich (Moscow, Russia). Furosemide (Sanofi S.A., France) was used as a positive control in the diuretic activity assessment.

Plant material

Plant material of V. nigrum was collected on the collector area of Perm State Pharmaceutical Academy, Perm Region, the Russian Federation. This plant was identified according to Russian Flowering Plant Database by the expert and voucher specimen (No. S0632) was deposited at the Pharmacognosy Department Herbarium of the Perm State Pharmaceutical Academy for future reference. The plant material was dried in a dark, dry place at room temperature until constant weight of the material is achieved.

Dry herb of Equisetum arvense L. (Equisetaceae) and dry leaves of Vaccinium vitis-idaea L. (Ericaceae) were purchased from Health-joint stock Co. (PN 003011/02 and PN 001008/01, respectively) and their extracts were used as reference natural diuretic drugs.

Extract preparation

The dry V. nigrum extracts of aerial parts viz. herb (VnH), leaves (VnL), stems (VnS), flowers (VnF), and roots (VnR) were prepared by repercolation according to the previously reported method (Kalinina et al., 2012). Briefly, raw material of V. nigrum crushed to particles less than 1 mm was extracted using a complete cycle repercolation in a three percolator battery. The plant material was charged in the percolators in decreasing amounts (50:30:20 ratio) and was percolated for 8 h using 60% ethanol as a solvent. After collection of the ethanol extract, the solvent was evaporated under vacuum using rotary evaporator affording the dry extracts, which were used for further analysis and pharmacological evaluation.

The dry extract of the V. vitis-idaea (VvL) leaves was obtained by the standard method (Bobkov & Babayan, 1991a). Briefly, 10 g of dry plant material was boiled in 200 ml of water. After 30 min, the extract was collected by filtration. The solvent was evaporated under vacuum using rotary evaporator to give 2.4 g of the dry extract.

The dry extract of the E. arvense (EaH) herb was obtained by the standard method (Bobkov & Babayan, 1991b). Briefly, 10 g of dry plant material were mixed with 200 ml of hot water. After the initial 15 min infusion, the mixture was boiled for 20 min and the extract was collected after filtration. The solvent was evaporated under vacuum using a rotary evaporator to give 1.9 g of the dry extract.

Preparation of extract fractions

The extraction and fractionation were carried out according to the scheme outlined in Figure 1. The dry herb of V. nigrum (305 g) crushed to particles less than 5 mm was extracted with 1500 ml of 70% ethanol by heating the mixture under reflux for 5 h. The same procedure was repeated using 800 ml of 40% ethanol followed by the same amount of water. These three extracts were combined and concentrated to the volume of 200 ml under reduced pressure. The lipophilic compounds and chlorophyll were removed by liquid--liquid extraction (LLE) with chloroform (3 × 250 ml). The remaining aqueous fraction was mixed with small amount of polyamide sorbent (50–160 μm, Fluka, Germany) and dried under vacuum prior to loading into the chromatographic column. Identification of the constituents was performed using co-chromatography with authentic standard samples of hesperidin, quercetin, luteolin, aucubine, chlorogenic and caffeic acid. The presence of flavonoids, iridoids or phenolic acids in fractions was assessed on the basis of color reaction tests.

Figure 1. Scheme of V. nigrum extract fractionation.

The column chromatography was performed on polyamide sorbent collecting 125 of the 100 ml fractions (column length 15 cm, diameter 5 cm). The elution was started with water and was continued until negative tests to iridoids (fractions 1 to 30) were obtained. The total volume of aqueous fractions was reduced by evaporation under vacuum to 200 ml and was then treated with 96% ethanol to precipitate polysaccharides, which were then removed by filtration. The filtrate was evaporated under vacuum providing the iridoid fraction (Ir). The development of the column chromatography was subsequently continued using 10% ethanol until negative tests to phenolic acids (fractions 31 to 55) were obtained. After evaporation of the solvent, the residue was recrystallized from ethyl acetate affording the phenolic acids fraction (PhAc). Then increasing concentrations of ethanol were applied for elution: fractions 56 to 80 were collected using 40% ethanol, fractions 81 to 111 using 80% ethanol and fractions 112–125 using 96% ethanol. The chromatographic development was continued until negative tests for flavonoids. After removal of the solvent using a rotary evaporator, three flavonoid fractions Fl1, Fl2 and Fl3 were obtained.

Extract standardization

The V. nigrum extracts were standardized on the basis of quantitative content of: flavonoids (expressed as hesperidin) using previously reported method (Kalinina & Petrichenko, 2012) and iridoids (aucubine) using the Grëger and Simchen method (1967). The composition of the V. nigrum extract fractions was determined chromatographically using hesperidin, quercetin and luteolin standards (Sigma-Aldrich).

Animals

Experiments were conducted on Swiss albino mice of both sexes (18 to 22 g) and male Wistar albino rats (250 to 300 g) housed and maintained under laboratory conditions at 25 °C, normal 12 h:12 h light/dark schedule with free access to water and food. The animals were allowed sufficient time to adapt to the laboratory environment before the experiments. The experiments were conducted in accordance to internationally accepted standard procedures for animals use and were approved by the Animal Ethic Committee of Perm State Pharmaceutical Academy (permission 2009-B-03).

Evaluation of acute toxicity

The acute toxicity of each extract was determined using Swiss albino mice of both sexes with body weight 18 to 22 g. The V. nigrum extracts and the fractions of the constituents were administered orally as aqueous solutions (0.5 ml) in increasing doses. The maximal dose used was 5000 mg/kg. After the treatment, animals were observed for possible mortality cases and behavioural changes for 72 h. LD₅₀ values were estimated according to reported methods (Prozorovsky et al., 1978).

Determination of diuretic activity

Rats were divided into 14 equal groups (n = 6) prior to oral administration of 5 ml of distilled water, reference drug or aqueous solutions of the same volume of the extracts. The negative control group received 5 ml of distilled water; the positive control groups were treated with the extracts of E. arvense (50 mg/kg), V. vitis-idaea (50 mg/kg) or furosemide (50 mg/kg) dissolved in distilled water. The experimental groups received the preparations (50 mg/kg) dissolved in distilled water. After the treatment, animals were placed in individual metabolic cages and were kept under observation. The urine was collected and its volume was measured after 3 and 6 h of the treatment.

Determination of urinary electrolytes

The concentrations of sodium and potassium ions in urine were determined using a Jenway Corp. model PSP7 flame photometer after dilution of urea with distilled water (1:2 and 1:20 for determination of sodium and potassium ions, respectively).

Data and statistical analysis

Data are expressed as mean ± standard error of mean (S.E.M.). The difference between the means of treated groups and control group was evaluated by the Student’s unpaired t-test and a probability level lower than 0.05 (p < 0.05) was considered as statistically significant.

Results

Plant extract

To identify organs of the highest deposition of active compound(s), the extraction was performed from different parts of the plant. The yield and results of the chemical standardization for the V. nigrum extracts are shown in Table 1.

Table 1. Extracts of Verbascum nigrum L., their yields and content of principal components.

		Principal components
Extract	Yield (%)^a	Flavonoids (%)^b	Iridoids (%)^c
VnH	27.2	8.2 ± 0.4	1.2 ± 0.01
VnL	24.5	6.5 ± 0.2	1.1 ± 0.02
VnS	30.1	10.2 ± 0.7	2.3 ± 0.01
VnF	20.1	5.5 ± 0.4	2.1 ± 0.01
VnR	22.8	3.8 ± 0.5	0.5 ± 0.01

Values expressed as mean ± S.E.M. (n = 3).

^aYield of dried extracts on dry plant basis.

^bFlavonoid content in the extracts expressed as hesperidin equivalents.

^cIridoid content in the extracts expressed as aucubin equivalents.

The yields of extracts from V. nigrum were within 20.1 to 30.1%. The mullein stems were found to be the richest part of the plant in terms of the flavonoid and iridoid contents. Similar iridoid quantities were present in the mullein flowers. However, low content of iridoids in the extracts can be attributed to the type of solvent used for the extraction (60% ethanol) and does not represent the actual level of iridoids in the mullein plant material.

Preparation of extract fractions

The extract for the fractionation was prepared using a series of solvents of different polarity to ensure maximal extraction of mullein constituents. The extract was fractionated resulting in isolation of three groups of flavonoids, one group of iridoids and a group of phenolic acids. The yields of each fraction with respect to the dry material are shown in Table 2.

Table 2. Fractions of the Verbascum nigrum L. extract.

Fraction	Yields (g)	Yields (%)^a
Flavonoids (EtOH 40%)^b (Fl1)	5.5 ± 0.27	1.8 ± 0.07
Flavonoids (EtOH 80%)^b (Fl2)	4.0 ± 0.2	1.3 ± 0.07
Flavonoids (EtOH 96%)^b (Fl3)	1.5 ± 0.08	0.5 ± 0.03
Flavonoids total	11.0 ± 0.55	3.6 ± 0.18
Iridoids (Ir)	8.0 ± 0.4	2.7 ± 0.14
Phenolic acids (PhAc)	0.1 ± 0.01	0.03 ± 0.003

Values expressed as mean ± S.E.M. (n = 3).

^aYields of dried fractions on basis of dry plant material used for the extraction.

^bEluent used for the fraction preparation.

The flavonoid fractions were the major components of the extract. They appeared as a light brown crystalline powder with bitter taste soluble in water. Iridoids were effectively isolated yielding a fraction soluble in water. It was a brown colored, amorphous powder having bitter taste. The fraction of phenolic acids was isolated as a yellowish powder with bitter taste soluble in water.

The major components of the flavonoid fraction obtained after the elution with 40% ethanol were hesperidin, quercetin and luteolin (54% expressed as hesperidin equivalents). Neither quercetin nor luteolin were detected in the fractions collected upon the elution with ethanol of higher concentrations. The main component of these fractions was hesperidin. The total flavonoid content in the fractions obtained after elution with 80 and 96% ethanol was 89 and 80%, respectively (expressed as hesperidin equivalents). The fraction of iridoids was mainly represented by aucubine with total iridoid content of 64% expressed as aucubine equivalents. Chlorogenic and caffeic acids were identified in the fraction of phenolic acids.

Acute toxicity

The V. nigrum extracts demonstrated a low toxicity profile. No effect on mouse behavioral responses and no mortality cases were observed during the 72 h period after the administration of the doses up to 5000 mg/kg (p.o.). The same lower toxicity was observed for the iridoid fraction of the V. nigrum extract, but the fractions of phenolic acids and flavonoids were more toxic with LD₅₀ values of 1030 and 870 mg/kg (p.o.), respectively.

Diuretic activity

Extracts from aerial parts of the plant were found to possess diuretic activity (Table 3). Extracts from all parts of V. nigrum, except the one obtained from roots, significantly increased urine excretion. The extract from stems demonstrated more pronounced diuretic effect when compared with extracts from other parts of the plant. Level of diuretic activity of this extract was also significantly higher (57%, p < 0.01) than that of the E. arvense extract. The maximum output of urine was observed during the first three hours after the treatment. Furthermore, none of the V. nigrum extracts increased urine excretion in the 3 to 6 h intervals after the treatment.

Table 3. Effect of oral administration (50 mg/kg) of extracts of Verbascum nigrum L. and reference extracts of Vaccinium vitis-idaea L. and Equisetum arvense L. and furosemide on urinary excretion volume.

	Urine volume (ml)
Treatment	3 h	6 h	Total	Urine excretion (ml/kg BW)^a	Diuretic index^b
Control	0.8 ± 0.08	0.5 ± 0.04	1.3 ± 0.2	5.2 ± 1.4	–
VnH	3.1 ± 0.2***	0.4 ± 0.1	3.5 ± 0.7**	12.9 ± 2.4*	2.5
VnL	2.6 ± 0.3***	0.2 ± 0.1*	2.8 ± 0.1***	9.3 ± 0.5*	1.8
VnS	3.5 ± 0.2***	0.6 ± 0.3	4.1 ± 0.2***	14.6 ± 0.8***	2.8
VnF	2.0 ± 0.5*	0.6 ± 0.2	2.6 ± 0.5*	8.7 ± 2.2	1.7
VnR	0.5 ± 0.1*	0.2 ± 0.05***	0.7 ± 0.2	2.3 ± 0.5	0.4
EaH	2.2 ± 0.1***	0.5 ± 0.2	2.6 ± 0.1***	9.3 ± 0.7*	1.8
VvL	3.8 ± 0.2***	1.0 ± 0.5	4.8 ± 0.3***	17.8 ± 1.1***	3.4
Furosemide	7.3 ± 0.5***	2.1 ± 0.4*	9.4 ± 0.3***	37.6 ± 1.9***	7.2

Values expressed as mean ± S.E.M. (n = 6).

*p < 0.05; **p < 0.01; ***p < 0.001 compared to control group using Student’s t-test.

^aUrine excretion on basis of average body weight in the groups.

^bDiuretics index = (urinary excretion of treated group)/(urinary excretion of control group).

To identify group(s) of the chemical constituents of mullein responsible for the diuretic activity, five fractions viz. flavonoids (Fl1, Fl2 and Fl3), iridoids (Ir) and phenolic acids (PhAc) were prepared for pharmacological evaluation. All flavonoid fractions demonstrated significant diuretic activity exceeding effects of the reference extracts of E. arvense and V. vitis-idaea (Table 4). Fractions Fl2 and Fl3 were practically equipotent and nearly as active as a standard diuretic drug furosemide in the same dose. The increase of diuresis induced by these fractions (Fl2 and Fl3) was 4.4-times higher than the diuretic action of the E. arvense extract and 2-times higher than the increase of urine excretion caused by the V. vitis-idaea extract. No apparent diuretic activity was observed upon treatment of the animals with the fractions of iridoids and phenolic acids.

Table 4. Effect of oral administration (50 mg/kg) of extract fractions of Verbascum nigrum L., and reference extracts of Vaccinium vitis-idaea L. and Equisetum arvense L. and furosemide on urinary excretion volume.

	Urine volume (ml)
Treatment	3 h	6 h	Total	Urine excretion (ml/kg BW)^a	Diuretic index^b
Control	0.8 ± 0.08	0.5 ± 0.04	1.3 ± 0.2	5.2 ± 1.4	–
Fl 1	4.5 ± 1.2*	2.1 ± 0.7*	6.6 ± 1.5***	26.5 ± 3.1***	5.1
Fl 2	4.8 ± 1.7*	2.4 ± 0.9	7.2 ± 1.9*	35.9 ± 2.1***	6.9
Fl 3	5.3 ± 1.4**	1.8 ± 0.7	7.1 ± 1.2**	35.4 ± 3.9***	6.8
Ir	0.7 ± 0.1	0.5 ± 0.1	1.3 ± 0.4	5.2 ± 1.8	1
PhAc	1.0 ± 0.2	0.8 ± 0.2	1.8 ± 0.5	7.3 ± 1.9	1.4
EaH	2.2 ± 0.1***	0.5 ± 0.2	2.6 ± 0.1***	9.3 ± 0.7*	1.8
VvL	3.8 ± 0.2***	1.0 ± 0.5	4.8 ± 0.3***	17.8 ± 1.1***	3.4
Furosemide	7.3 ± 0.5***	2.1 ± 0.4*	9.4 ± 0.3***	37.6 ± 1.9***	7.2

Values expressed as mean ± S.E.M. (n = 6).

*p < 0.05; **p < 0.01; ***p < 0.001 compared to control group using Student’s t-test.

^aUrine excretion on basis of average body weight in the groups.

^bDiuretics index = (urinary excretion of treated group)/(urinary excretion of control group).

Electrolyte excretion

Table 5 shows the ionic excretion observed after oral administration of the extracts. The sodium excretion after the treatment of animals with the extracts from stems of V. nigrum was four times higher than the control group level for both intervals (3 and 6 h) of urine collection. The extract from leaves and to lesser extent the flower extract also possessed natriuretic activity. Logically, the herb extract was found to possess this activity as well. The extract from roots demonstrated no increase in sodium excretion at any of these intervals. The data showed a remarkable parallelism among sodium and urine excretion for first 3 h after the treatment. Surprisingly, no such parallelism was observed for the following 3 h. In general, all diuretically active mullein extracts also possessed kaliuretic effect. The treatment of animals with the stem extract led to the highest level of potassium excretion (K⁺ 3.8) when compared with the use of the extracts from other parts of the plant.

Table 5. Effect of oral administration (50 mg/kg) of extracts of Verbascum nigrum L. and reference extracts of Vaccinium vitis-idaea L. and Equisetum arvense L. and furosemide on urinary electrolyte excretion.

	Na ^+				K^+
Treatment	3 h (mEq/100 g)	6 h (mEq/100 g)	Total (mEq/100 g)	S.i. Na^+	3 h (mEq/100 g)	6 h (mEq/100 g)	Total (mEq/100 g)	S.i. K^+	Na^+/K^+
Control	0.51 ± 0.04	0.42 ± 0.04	0.93 ± 0.02	–	0.14 ± 0.01	0.10 ± 0.03	0.24 ± 0.01	–	3.88
VnH	2.01 ± 0.2***	1.51 ± 0.02***	3.52 ± 0.04***	3.8	0.57 ± 0.05***	0.28 ± 0.02***	0.85 ± 0.01***	3.5	4.14
VnL	1.30 ± 0.02***	0.84 ± 0.03***	2.14 ± 0.01***	2.3	0.49 ± 0.04***	0.17 ± 0.05	0.66 ± 0.01***	2.8	3.24
VnS	2.23 ± 0.11***	1.64 ± 0.07***	3.87 ± 0.02***	4.1	0.59 ± 0.03***	0.32 ± 0.05**	0.91 ± 0.01***	3.8	4.25
VnF	0.91 ± 0.03***	0.60 ± 0.02**	1.51 ± 0.01***	1.6	0.23 ± 0.05	0.15 ± 0.07	0.38 ± 0.02***	1.6	3.97
VnR	0.40 ± 0.02	0.42 ± 0.08	0.82 ± 0.02***	0.9	0.09 ± 0.01**	0.08 ± 0.01	0.17 ± 0.01**	0.7	4.82
EaH	0.80 ± 0.02***	0.08 ± 0.01***	0.88 ± 0.01**	0.9	0.21 ± 0.01**	0.10 ± 0.01	0.31 ± 0.01**	1.3	2.84
VvL	2.63 ± 0.04***	0.52 ± 0.02*	3.15 ± 0.02***	3.4	0.79 ± 0.09***	0.09 ± 0.01	0.88 ± 0.02***	3.7	3.58
Furosemide	0.74 ± 0.04**	0.77 ± 0.01**	1.51 ± 0.02***	1.6	0.21 ± 0.03*	0.24 ± 0.05*	0.45 ± 0.02***	1.9	3.35

Values are expressed as mean ± S.E.M. (n = 6). S.i. = Saluretic index = (Ion excretion in treated group)/(Ion excretion in control group).

*p < 0.05; **p < 0.01; ***p < 0.001 compared to control group using Student’s t-test.

The experiments carried out with the fractions of different groups of constituents demonstrated similar results for both sodium and potassium excretion (Table 6). Thus, only flavonoid fractions were found to possess saluretic activity. The hesperidin rich fractions Fl2 and Fl3 increased sodium excretion 4.5-times when compared with control. The excretion of potassium by the same fractions was about 5-times higher than that of the control (S.i. K⁺ 5.4 and 4.9, respectively). No increase in the ion excretion was observed after treatment of animals with the fractions of iridoids and phenolic acids.

Table 6. Effect of oral administration (50 mg/kg) of extract fractions of Verbascum nigrum L., and reference extracts of Vaccinium vitis-idaea L. and Equisetum arvense L. and furosemide on urinary electrolyte excretion.

	Na^+				K^+
Treatment	3 h (mEq/100 g)	6 h (mEq/100 g)	Total (mEq/100 g)	S.i. Na^+	3 h (mEq/100 g)	6 h (mEq/100 g)	Total (mEq/100 g)	S.i. K^+	Na^+/K^+
Control	0.51 ± 0.04	0.42 ± 0.04	0.93 ± 0.02	–	0.14 ± 0.01	0.10 ± 0.03	0.24 ± 0.01	–	3.88
Fl 1	2.39 ± 0.2***	1.51 ± 0.06**	3.90 ± 0.04***	4.2	0.72 ± 0.06***	0.43 ± 0.06***	1.15 ± 0.02***	4.8	3.39
Fl 2	2.92 ± 0.1***	1.30 ± 0.07***	4.22 ± 0.02***	4.5	0.81 ± 0.06***	0.49 ± 0.04***	1.30 ± 0.01***	5.4	3.25
Fl 3	3.03 ± 0.2***	1.22 ± 0.05***	4.25 ± 0.04***	4.5	0.73 ± 0.08***	0.45 ± 0.04***	1.18 ± 0.02***	4.9	3.60
Ir	0.5 ± 0.03	0.3 ± 0.02*	0.80 ± 0.01**	0.9	0.09 ± 0.01***	0.09 ± 0.01	0.18 ± 0.01**	0.8	4.44
PhAc	0.49 ± 0.01	0.42 ± 0.04	0.91 ± 0.01	1.0	0.12 ± 0.05	0.11 ± 0.01	0.23 ± 0.01	1.0	3.95
EaH	0.80 ± 0.02***	0.08 ± 0.01***	0.88 ± 0.01**	0.9	0.21 ± 0.01**	0.10 ± 0.01	0.31 ± 0.01**	1.3	2.84
VvL	2.63 ± 0.04***	0.52 ± 0.02*	3.15 ± 0.02***	3.4	0.79 ± 0.09***	0.09 ± 0.01	0.88 ± 0.02***	3.7	3.58
Furosemide	0.74 ± 0.04**	0.77 ± 0.01**	1.51 ± 0.02***	1.6	0.21 ± 0.03*	0.24 ± 0.05*	0.45 ± 0.02***	1.9	3.35

Values are expressed as mean ± S.E.M. (n = 6). S.i. = Saluretic index = (Ion excretion in treated group)/(Ion excretion in control group).

*p < 0.05; **p < 0.01; ***p < 0.001 compared to control group using Student’s t-test.

Discussion

There are a number of plants with confirmed diuretic activity (Wright et al., 2007). The most explored plants possessing this pharmacological action include Equisetum arvense L. (Equisetaceae) (Knöss, 2008), Vaccinium vitis-idaea L. (Ericaceae) (Stepanova & Stusenko, 2008), Orthosiphon stamineus Benth. (Lamiaceae) (Englert & Harnischfeger, 1992), Urtica dioica L. (Urticaceae) (Koch, 2001), Arctosеaphylos uva-ursi L. (Ericaceae) (Beaux et al., 1999), Boerhaavia verticillata Poir. (Nyctaginaceae) (Bajpai & Ojha, 2000) and Colliguaja integerrima Gilles and Hook (Euphorbiaceae) (Alvarez et al., 2009). We prepare extracts from V. nigrum and explored their effects on diuresis.

Identification of the plant organs with the highest content of compounds responsible for the bioactivity is an important part of the rationalization process for optimal use of plant material. Therefore, we carried out the extraction from the herb as well as individual parts of the plants and compared their biological activity. In folk medicine, no standard method exists for preparation of the remedies from V. nigrum. It was reported (Kalinina et al., 2012) that optimal solvent for the extraction of majority of the bioactive constituents from V. nigrum was 60% ethanol. Therefore, we used this solvent for the preparation of the extracts for pharmacological evaluation and the optimal parameters established during the same work (Kalinina et al., 2012).

The diuretic activity study of the extracts obtained from different parts of V. nigrum demonstrated that the extract from stem was more potent than extracts from other parts of the plant. This extract tripled the urine output compared to control and was more active than the E. arvense extract.

The clear relationship was observed between the flavonoid content and ability of the extracts to increase urine excretion. The extract from stems of V. nigrum, which possessed the highest diuretic activity (D.i. 2.8), was found to be the most flavonoid rich (10.2%) extract of this plant. The lowest content of flavonoids was identified in the extract from roots, which showed no diuretic activity. The iridoids content in the extracts was much lower than the flavonoid content as iridoids in general were less extractable by ethanol. Due to the low content of iridoids in these extracts, estimation of their contribution to the diuretic activity of extracts seemed to be problematic without additional experiments.

It has been well established that various types of constituents, such as saponins, organic acids and phenolic compounds may dictate diuretic activity of extracts. Diuretic effect of flavonoids (Graefe & Veit, 1999; Makarov, 1972) and their glycosides (Borkowski, 1960), e.g., luteolin (Compaore et al., 2011), hesperidin (Galati et al., 1996; Garg et al., 2001) and isoquercitin (Gasparotto et al., 2011) have been demonstrated in a number of reports. High diuretic potency of the V. vitis-idaea extract can be reasonably attributed to substantial content of arbutin in this extract.

In order to unambiguously identify group of constituents responsible for the diuretic effect of the V. nigrum extracts, we isolated fractions of polar compounds, which were more likely to possess this biological activity. Thus, three fractions of flavonoids, a fraction of iridoids and a fraction of phenolic acids were obtained using column chromatography on polyamide sorbent. It was observed that elution with ethanol of higher concentration increased content of hesperinin in the flavonoid fractions. These fractions demonstrated the highest level of diuretic activity as well as natriuretic and kaliuretic effects. A decrease of the hesperidin content in the flavonoid fraction collected after elution with 40% ethanol was observed that probably translated into less pronounced pharmacological activity. The iridoid fraction was pharmacologically inert in all the experiments we carried out. Some increase in the diuresis was observed after treatment of animals with the phenolic acid fraction; however, the difference with the control group was not statistically significant. Therefore, the diuretic activity of the V. nigrum extracts can be attributed to flavonoids and to greater extent to hesperidin content. This fact perfectly correlates with the strong diuretic action of the flavonoid rich extracts from stems and herb of V. nigrum. Similarity in the pharmacological profile of the V. nigrum extracts and the preparations from E. arvense and V. vitis-idaea can also be explained on the basis of the nature of chemical constituents present. Both E. arvense and V. vitis-idaea increase the urine and ion excretion and these effects are attributed to their flavonoids, quercetin and arbutin, respectively.

Though we identified that the diuretic activity of the V. nigrum extracts was due to the flavonoids, the mechanism of pharmacological effects is not very clear. An initial vasodilatation (Stanic & Samarzija, 1993) may at least partially contribute to the increase of urine output. The vasodilatation might also explain the earlier reported hypotensive effect of the V. nigrum extracts (Kalinina et al., 2011). Both diuretic and hypotensive activities were also found for hesperidin (Galati et al., 1996) that confirm our assumption that this flavonoid was among the constituents responsible for the diuretic activity of V. nigrum.

It should also be noted that the most diuretically active flavonoid fractions demonstrated the higher acute toxicity level.

Conclusion

This study experimentally proved the diuretic activity of V. nigrum, which is used in traditional medicine. The plant also demonstrated saluretic activity. It was established that the extracts from stems and herb of V. nigrum were the most potent. The flavonoid hesperidin appeared to be the main contributor to the biological activity of the plant that was further confirmed by the investigations of the polar compound fractions.

Declaration of interest

The project was in part financially supported by Russian Ministry of Education and Science. The authors report no declarations of interest.

Notes

#Dedicated to the memory of Professor V.M. Petrichenko, our mentor, colleague and friend.