Diuretic activity of squamate mistletoe, Viscum angulatum

Abstract

Viscum angulatum Heyne ex DC (Viscaceae) is a leafless hemiparasitic shrub traditionally used in Asian countries for the treatment of hypertension. In the present study, the methanol extract of the whole plant was examined for diuretic activity in rats. The activity was evaluated using parameters such as urine volume after 5, 19, and 24 h and urine sodium, potassium, and chloride concentrations. The extract was further scrutinized for polyphenolic compounds and triterpenoids. The extract demonstrated a significant increase in and dose-dependent effect on urine excretion volume in comparison to the normal group in the tested range of 100–400 mg/kg. The extract demonstrated comparable saluretic and higher natriuretic activity (Na⁺/K⁺) compared to the furosemide treated group. However, the Cl⁻/Na⁺ + K⁺ ratio, which indicates carbonic anhydrase mediated activity, remained unaffected. HPLC and quantitative analysis of the extract revealed that polyphenolic compounds and the triterpenoid, oleanolic acid, are the major phytochemicals, and are proposed to be responsible for the observed diuretic effect. Oleanolic acid has been reported to possess diuretic activity with significant potassium loss in rats. In contrast to pure oleanolic acid, the extract demonstrated a valuable potassium-sparing effect. This suggests modulation of the diuretic effect of oleanolic acid by polyphenolics present in the extract. The observed dose-dependent potassium-sparing diuretic effect is a hereto unreported property of this plant.

Keywords::

• Diuretic
• oleanolic acid
• potassium sparing
• polyphenolics
• Viscum angulatum

Introduction

The mistletoe species (hemiparasitic plants of families Loranthaceae and Viscaceae) have long been used as medicinal herbs. The mistletoe Viscum angulatum Heyne ex DC (Viscaceae) is a leafless hemiparasitic shrub endemic to Asian countries (Kirkup et al., 2000). The whole plant is traditionally used in China to treat hypertension, atherosclerosis, rheumatism, neuralgia, and arthritis (Chiu & Chang, 1986). Previous phytochemical work revealed that flavonoids and pentacyclic triterpenoids are the major secondary metabolites of this species (Rastogi & Mehrotra, 1993). In addition, phenolic glycosides were also reported from the plant (Lin et al., 2002).

Ethnomedicinal review of some 52 mistletoe species revealed that these plants are frequently used in the treatment of hypertension. Increased diuresis has also been reported during mistletoe infusion used for the treatment of hypertension as a folk remedy (Deliorman et al., 2000). In a preliminary study, referring to the current study, conducted to investigate the effect of the methanol extract on normal blood pressure in rats, we observed that besides significant hypotensive effects, the extract also exerted diuresis in the rats. The present study was thus designed to determine the effect of the methanol extract of V. angulatum on urinary excretion of water, sodium, potassium, and chloride in rats, and to perform some studies to determine the phytochemicals responsible for the observed effects.

Materials and methods

Plant material and extract preparation

The whole plant of V. angulatum parasitic on Randia dumetorum Lamb. (Rubiaceae) was collected from the tropical evergreen forest of Western Ghat (16° 41′ 60N latitude, 74° 13′ 0E longitude, and 1000 m altitude) in November 2007. The plant specimen (Voucher no. RBJ 1) was authenticated by Dr. S. C. Majumdar of the Botanical Survey of India, Pune. The air-dried whole plant material (900 g) was extracted with methanol (3 × 2.5 L) by cold maceration for 24 h, and pooled extracts were concentrated on a rotary vacuum evaporator. The yield was 6.53%, w/w.

Animals

Twelve-week-old male Wistar rats weighing 170–175 g and Swiss albino mice of 5–7 weeks age weighing 20–25 g were obtained from registered vendors. The animals were housed in separate cages containing four animals per cage, and were maintained under control conditions of 22 ± 2°C, with a 12 h light/dark cycle. Food and water were provided ad libitum till the day prior to the study. Ethical clearance was obtained from the Institutional Animal Ethical Committee (Approval no. 652/02/C/CPCSEA).

Acute toxicity study

The toxicity study was performed as per OECD (Organisation for Economic Co-operation and Development) guideline number 425 (Adeneye et al., 2006). A limit test was performed initially. Swiss albino mice weighing 20–25 g were used in the toxicity study. Six mice were serially administered with a 2000 mg/kg dose of extract prepared in water as recommended in the guideline. After dose administration, each animal was observed after every hour for signs of toxicity and abnormality in behavior up to 48 h, followed by daily observations for toxicity and mortality up to 14 days. Body weight of the animal was recorded on every third day. On the 14th day post-dosing, all the mice were sacrificed and processed for gross necropsy.

Diuretic activity

The assessment of diuretic activity was performed using the method reported by Lipschitz et al. (1943). Each treatment group contained eight male albino rats. The rats were fasted and deprived of water for 18 h before the experiment. Screening of diuretic activity was started in the morning between 7.00 and 8.00 a.m. on the subsequent day. Each rat received an oral load of 2.5 mL of 0.9% NaCl/100 g of body weight (Wiebelhaus et al., 1965). The extract dissolved in water was administered at doses of 100, 200, and 400 mg/kg to different groups. Care was taken to keep the volume of this dose equal in all rats. Rats in the control group were given an equal amount of water in addition to saline as described above. As a standard drug, furosemide (Aventis, India) at the dose of 10 mg/kg was given orally. Immediately after dosing, animals were placed in metabolic cages (one in each cage), specially designed to separate the urine and feces, and kept at room temperature. The parameters noted for individual rats were: urine volume after 5, 19, and 24 h; and urine sodium, potassium, and chloride concentrations. Sodium and potassium concentrations were measured using a flame photometer (Systronic-128; India) (Agunu et al., 2005) and chloride estimation was performed with a commercially available kit (Accurex Biomedical, India) using the method reported by Schoenfeld and Lewellan (1964). The sum of Na⁺ and Cl⁻ excretion was calculated as a parameter of saluretic activity. The ratio Na⁺/K⁺ was calculated for natriuretic activity and the ratio Cl⁻/Na⁺ + K⁺ (ion quotient) was calculated to determine carbonic anhydrase inhibition.

Estimation of sodium and potassium in plant extract

The concentration of potassium and sodium in the extract solution (1 mg/mL) prepared in distilled water was determined using a flame photometer (Systronic-128; India).

Total phenolic, flavonoid, and proanthocyanidin contents

The total phenolic content of the methanol extract was determined using Folin–Ciocalteu reagent and expressed as gallic acid equivalents (Singleton & Rossi, 1965). The flavonoid and proanthocyanidin contents were determined using methods previously reported (Porter, 1989; Liu et al., 2005) and expressed as quercetin and epicatechin equivalents, respectively. Gallic acid, quercetin, and epicatechin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All quantifications were performed in triplicate using a Shimadzu UV-1601 spectrophotometer.

HPLC analysis of triterpenoids

The qualitative analysis of triterpenoids in the methanol extract was performed using a PerkinElmer 200 series high performance liquid chromatography (HPLC) system. Chromatographic separation was performed on an Exsil ODS column (250 cm × 4 mm, 5 µm particle size) in isocratic mode with acetonitrile:water (85:15, v/v) as the solvent system. The temperature of the column was kept constant at 30°C and the mobile phase was delivered at a flow rate of 1.0 mL/min; the elution was monitored by a photodiode array detector set at 215 nm. The chromatograms were processed using Turbochrom software. The extract, 10 mg, was sampled, dissolved in methanol, and analyzed. Phytochemical markers of oleanolic acid and betulinic acid were obtained from Sigma Chemical Co., USA. Acetonitrile, water, and methanol (HPLC grade) were purchased from Merck Ltd., India.

Statistical analysis

The data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test, using GraphPad Prism 4.0 software. The level of significance was determined in comparison with the control group.

Results

In the acute toxicity study of the extract in mice as per OECD guideline 425, none of the six animals treated with 2000 mg/kg, p.o. dose of extract died or revealed any observable neurobehavioral effects during the observation period of 14 days. In the necropsy following euthanasia, no significant alterations in the histology of vital organs were observed. Hence, as per OECD guideline number 425, this extract cannot be categorized under the “Global Harmonization System.” Due to lack of observable toxicity at the 2000 mg/kg dose, no further determination of the actual LD₅₀ was carried out. The observed least toxicity is consistent with the use of the plant as a food source by the tribal community of Karnataka (Krishnamani & Kumar, 2000).

The results of the diuretic study indicate that the extract had a significant effect on the urine excretion volume in comparison to the normal group in the tested range of 100–400 mg/kg (Table 1). The results also indicate that the extract had a dose-dependent effect on urine output, and the dose of 400 mg/kg at 24 h exerted a diuretic index of about 76% of that of the furosemide treated group. In contrast to the acute diuretic effect of furosemide, which is limited to 5 h, the extract demonstrated sub-acute and consistent diuresis up to 24 h. The results of urine electrolyte analysis indicate that potassium loss was significantly higher in the furosemide treated group in comparison to the control group (p < 0.01) (Table 2). However, extract treated groups showed meager potassium loss even at the higher dose of 400 mg/kg, as compared to the control group. Obviously the higher natriuretic activity (Na⁺/K⁺) observed at all doses of extract suggests a potassium-sparing effect (Table 2). The group treated with 400 mg/kg of extract showed comparable saluretic effect (Na⁺ + Cl⁻) to that of the furosemide treated group (Table 2). However, the ion quotient Cl⁻/Na⁺ + K⁺ was less influenced by extract treatment in comparison to the furosemide treated group.

Table 1.  Effect of methanol extract of Viscum angulatum (VA) on urine volume at 5 h, 19 h, and 24 h.

Group	Urine volume (mL)			Diuretic index^a
	5 h	19 h	24 h	5 h	19 h	24 h
Control	0.70 ± 0.27	2.57 ± 0.42	3.27 ± 0.59	–	–	–
Furosemide, 10 mg/kg	10.07 ± 0.83**	1.67 ± 0.51	11.74 ± 0.97**	14.38	0.65	3.60
VA, 100 mg/kg	2.58 ± 0.22*	3.28 ± 0.40	5.86 ± 0.63	3.68	1.28	1.80
VA, 200 mg/kg	2.77 ± 0.31**	4.57 ± 0.61*	7.34 ± 0.91**	3.95	1.78	2.25
VA, 400 mg/kg	3.90 ± 0.20**	5.13 ± 0.60**	9.03 ± 0.51**	5.57	2.00	2.76

Data are expressed as mean ± SEM, n = 8. Data were analyzed using one-way ANOVA followed by Dunnett’s test. *p < 0.05, **p < 0.01 compared to control group.

^aDiuretic index = volume in test group/volume in control group.

Table 2.  Effect of methanol extract of Viscum angulatum (VA) on urine sodium, potassium, and chloride concentration at 5 h.

Group	Urine electrolyte concentration (mmol/L)			Saluretic effect (Na + Cl)	Natriuretic effect (Na/K)	Cl/Na + K (ion quotient)
	Sodium	Potassium	Chloride
Control	19.67 ± 0.50	24.07 ± 2.96	87.00 ± 2.77	106.67	0.817	1.989
Furosemide, 10 mg/kg	66.03 ± 2.45**	97.10 ± 3.42**	154.10 ± 3.27**	170.13	0.680	0.945
VA, 100 mg/kg, p.o.	30.48 ± 2.83*	16.63 ± 1.95	93.71 ± 2.96	103.19	1.833	1.989
VA, 200 mg/kg, p.o.	54.63 ± 3.67**	31.70 ± 2.19	119.52 ± 3.01**	123.15	1.723	1.384
VA, 400 mg/kg, p.o.	74.10 ± 4.35**	33.60 ± 2.76	141.34 ± 3.49**	168.44	2.205	1.312

Data are expressed as mean ± SEM, n = 8. Data were analyzed using one-way ANOVA followed by Dunnett’s test. *p < 0.05, **p < 0.01 compared to control group.

The results for sodium and potassium content of the methanol extracts revealed a lower concentration of sodium as well as potassium in the methanol extract (sodium 23.69 mmol/L and potassium 11.36 mmol/L). Quantitative chemical analysis of the extract revealed the presence of polyphenolic compounds (284.1 ± 7.643 µg/mg); among them, proanthocyanidins constituted a major part (112.9 ± 5.939 µg/mg), followed by flavonoids (98.80 ± 6.445 µg/mg). HPLC analysis of the methanol extract revealed a preponderance of oleanolic acid along with a trace amount of betulinic acid (Figure 1).

Figure 1.  HPLC analysis of the methanol extract of Viscum angulatum (A) and reference compounds (B), betulinic acid (BA) and oleanolic acid (OA).

Discussion

This study reveals that the methanol extract of V. angulatum had significant and dose-dependant diuretic activity in rats. The results showed that the dose of 400 mg/kg of extract exerted comparable diuretic activity to that of furosemide (Table 1). In contrast to the acute diuretic effect of furosemide, which is largely terminated by 5 h, the extract demonstrated sub-acute but consistent diuresis. The urine volume measured at 19 h was lower in the furosemide treated group than in the control group, which suggests that the rats were depleted of water (Table 1). A similar observation of lower urine output in furosemide treated rats has been reported elsewhere (Ratnasooriya et al., 2004). It seems that the initial potent diuresis might have led to decreased output subsequently, since we did not provided the rats with water for the total duration of urine collection. Diuretic drugs, in general, decrease Na⁺ reabsorption in the kidney and other epithelial tissues, thereby leading to increased urine output. The most efficacious diuretic, furosemide, mediates diuresis by acting on sodium reabsorption in the ascending limb of the loop of Henle (Jackson, 1996). However, some unwanted effects are common with loop diuretics and are directly related to their renal action. Potassium loss resulting in low plasma K⁺ (hypokalemia) and metabolic alkalosis owing to H⁺ excretion are both very likely to occur (Rang et al., 2003). Consistent with these facts, the potassium loss was significantly higher in the furosemide treated group in comparison to the control group in our study. However, extract treated groups demonstrated insignificant potassium loss even at the higher dose of 400 mg/kg, compared to the control group (Table 2). Obviously the higher natriuretic activity (Na⁺/K⁺) observed at all doses of extract suggests a potassium-sparing diuretic effect. The extract also exhibited comparable saluretic activity (Na⁺ + Cl⁻) to that of the furosemide treated group. Furosemide is known to exert a modest effect as a carbonic anhydrase inhibitor (Stein et al., 1968; Radtke et al., 1972). Similarly, the extract demonstrated less influence on the ion quotient (Cl⁻/Na⁺ + K⁺) suggests no inhibition of carbonic anhydrase (Table 2). Lower potassium and sodium contents in the extract further support the conclusion of a potassium-sparing effect of the extract. Thus, V. angulatum is among the few plants that have potassium-sparing diuretic activity.

HPLC and quantitative analysis revealed that oleanolic acid and polyphenolic compounds were the major phytochemicals of the extract, and are proposed to be responsible for the observed diuretic effect. Previous work reported that oleanolic acid exerted diuretic activity with significant potassium loss in rats (Somova et al., 2003). On the other hand, the present study demonstrates higher natriuretic activity as well as a potassium-sparing effect of the extract even at the dose of 400 mg/kg. These observations, therefore, suggest that the diuretic effect of oleanolic acid, which is generally accompanied by significant potassium loss, was substantially reduced in the presence of polyphenolic compounds present in the methanol extract tested by us. This is an important observation, because the observed potassium-sparing effect appears to be the combined effect of the oleanolic acid and polyphenolics together. A wide variety of chemical compounds from both commercial diuretic drugs and plant secondary compounds can produce diuretic effects (Jackson, 1996; Dearing et al., 2001). However, most diuretic drugs typically act by a single mechanism (Jackson, 1996). All currently available diuretics perturb K⁺ homeostasis, and the discovery of a class of diuretic that is truly isokaluretic, i.e. neither increases nor decreases K⁺ excretion, is under extensive research (Jackson, 1996). Thus, the observed potassium-sparing diuretic activity of the oleanolic acid and polyphenolic compounds is an interesting example of a combination of diverse molecules having a favorable effect on a physiological process.

In conclusion, this study provides scientific evidence for diuretic activity of the methanol extract of V. angulatum. It further shows that the extract has significant and dose-dependent diuretic activity with a valuable potassium-sparing effect. A triterpenoid, oleanolic acid, and the polyphenolic compounds present in the methanol extract are proposed to be responsible for the observed diuretic effect. This effect further explains a possible mechanism of the antihypertensive property that is a common ethnomedicinal claim for many hemiparasites, including V. angulatum (Aleykutty et al., 1991, 1993; Deliorman et al., 2000).

Acknowledgements

The authors also acknowledge the help by taxonomists, Dr. C. B. Salunkhe and Dr. Milind Sardesai.

Declaration of interest

The authors are grateful to the All India Council for Technical Education, New Delhi for their generous grant (8023/BOR/RID/RPS/16/7/8).