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Effect of Bacopa monniera on liver and kidney toxicity in chronic use of opioids.

Abstract

In the present study, we investigated the protective effect of Bacopa monniera, an indigenous Ayurvedic medicinal plant in India, against morphine-induced liver and kidney toxicity in rats. Morphine intoxicated rats received 10-160mg/kg body weight of morphine hydrochloride intraperitoneally for 21 days. Bacopa monniera Extract (BME) pretreated rats were administered with BME (40mg/kg) orally once a day 2h before the injection of morphine for 21 days. Pretreatment with BME has shown to possess a significant protective effect against morphine-induced liver and kidney functions in terms of serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, alkaline phosphatase, lactate dehydrogenases and gamma-glutamyl transferase activities and urea, creatinine and uric acid level respectively. Histopathological changes of liver and kidney were also in accordance with the biochemical findings. The results of this study indicate that Bacopa monniera extract exerted a protection against morphine-induced liver and kidney toxicity.

[c] 2009 Elsevier GmbH. All rights reserved.

Keywords: Bacopa monniera; Morphine; Liver toxicity; Kidney toxicity

Introduction

The central role of liver and kidney in drug metabolism predisposes them to toxic injury. Every drug has been associated with hepatotoxity almost certainly due to the pivotal role of the liver in drug metabolism. Hepatic metabolism is first, and foremost, a mechanism that converts drugs and other compounds into products that are more easily excreted and that usually have a lower pharmacologic activity than the potent compound (Tolman 1998). A metabolite may have higher activity and/or greater toxicity than the original drug. Metabolites of the drugs that are excreted from kidneys may also cause cellular damage leading to kidney dysfunction (Singhal et al. 1998).

Opiates have been used clinically for more than a century (Way 1979). Morphine, a classic synthetic opiate, is still the main stay of treating acute pain from surgery, angina, myocardial infarction, and trauma (Macpherson 2000). Morphine, which is commonly used for the treatment of severe pain, is metabolized essentially in the liver, gastrointestinal tract and kidney (Stain-Texier et al. 1998; Pacifici et al. 1986).

Bacopa monniera (Linn) Wettst (Syn.Herpestis monniera (Linn) H.B& K family scrophulariaceae is a medicinal plant commonly known as Brahmi, have been used in the indigenous systems of medicine for the treatment of various nervous systems ailments such as insomnia, anxiety, epilepsy, hysteria, etc. (Nadkarni 1976). Preclinical and clinical studies have shown that Bacopa monniera improves memory and mental function (Roodenrys et al. 2002). The chronic effects of an extract of Bacopa monniera on cognitive function in human subject have been reported (Stough et al. 2001). Bacopa monniera also exhibits potent antioxidant (Tripathi et al. 1996), anticancer (Elangovan et al. 1995), antiulcer (Sairam et al. 2001), calcium antagonist (Dar and Channa 1999), vasodilatory (Channa et al. 2003), mast cell stabilizing (Samiulla et al. 2001), anti-inflammatory (Jain et al. 1994), anti stress (Chowdhuri et al. 2002), inhibitory effect on in vitro morphine withdrawal in guinea pig ileum (Sumathi et al. 2002), and anti-addictive properties (Sumathi et al. 2007). The nootropic activity of the extract has been attributed to the presence of two saponins, namely bacoside A and bacoside B, of which the former is the more important (Singh and Dhawan 1997). The present study was designed to explore the protective effect of Bacopa monniera against morphine-induced liver and kidney toxicity in rats.

Materials and methods

Chemicals

Morphine hydrochloride used in the present study was obtained from Moti and company Ltd., Chennai, Tamil Nadu, India. All other reagents used were analytical grade, and obtained from Himedia, India.

Preparation of plant extract

The plant Bacopa monniera was collected in and around Chennai, India and authenticated by Dr. P. Brindha, Central Research Institute (Siddha), Chennai, India. The shade dried and coarsely powdered whole plant material (1 kg) was extracted with 90% ethanol in the room temperature (48 h). The extract was filtered and distilled on a water bath to obtain a dark green syrupy mass. It was finally dried in vacuo (yield 52 g).

Animals

Adult male albino rats of wistar strain (120-200 g) were used for the present study. They were acclimatized to the laboratory condition cycles and maintained under 12h light/dark cycle at 25[+ or -]2[degrees]C. The experiments were carried out in accordance with the guidelines provided by the Institutional Animal Ethical Committee.

Experimental design

Adult male albino rats of the Wistar strain weighing 200-250g were maintained at constant temperature and light cycle with food and water ad libitum. After an acclimatization of 7 days, the rats were divided into four groups of six each. Group I rats received normal saline and served as a control. Group II, III and IV rats were treated with morphine hydrochloride, BME + morphine and BME alone respectively. While the dose of morphine hydrochloride was 10-160 mg/kg body weight/day i.p, for 21 days, that of BME was 40mg/kg body weight/day orally 2 h before the administration of morphine hydrochloride. The experiment was carried out for 21 days. On the last day the animals were sacrificed by cervical decapitation. The blood samples were allowed to clot for 30-40 min, serum was separated by centrifugation at 3000 rpm for 15 min at 37[degrees]C and used for various biochemical parameters.

HPLC-finger print analysis of Bacopa monniera extract

The following tabular column shows the exact data of the apparatus, column, solvent gradient, injection volume, detection wavelength and flow rate to which the Bacopa monniera extract was run for HPLC-finger print analysis.
HPLC system              Shimadzu HT2010 Chromatogrphic system with in
                         combination with Class LC 10A software & UV
                         detection

Column                   RP C-18 Luna phenomenex (250x4.6 mm)

Column oven temperature  25[degree]C

Mobile phase             A-0.25% orthophosphoric acid in water

                         B-Acetonitrile

Flow rate                1.5 ml

Injection volume         25.0[micro]l

Gradient                 Time   A. conc  B. conc

                         0.00   75       25

                         25.00  60       40

                         35.00  40       60

                         38.00  75       25

                         45     75.0     25

Detection wavelength     205 nm

Run time                 45 min

Sample preparation       Weigh accurately 500 mg extract to a 100 ml
                         volumetric flask dissolve in 50 ml methanol,
                         sonicate for 10-15 min. Cool then make up to
                         100 ml with methanol. Filter through 0.45
                         [mu]m membrane filter paper.


Biochemical analysis

SGOT and SGPT were assayed according to the method of Bergmeyer et al. (1976) and Bergmeyer (1980), respectively. The activity of ALP and LDH were measured by the method of King (1965a) and King (1965b), respectively. The activity of [gamma]-GT was assayed by the method of Rosalki and Rau (1972). The level of urea was estimated by the method of Natelson et al. (1951). The level of creatinine and uric acid were estimated according to the method of Owen et al. (1954) and Caraway (1963), respectively.

Histopathological examination

The liver and kidney specimens were fixed in 10% formalin for 24 h, and dehydrated in gradual ethanol (50-100%), cleared in xylene, and embedded in paraffin. Sections (4-5 [micro]m thick) were prepared and then stained with hemotoxylin and eosin (H-E) dye for photomicro-scopic observation.

Statistical analysis

Values are expressed as mean [+ or -]S.D (n = 6). The statistical significance of difference between the mean values were analysed by Student's t-test. A value of p<0.001 was regarded as statistically significant.

Results

Biochemical analysis

Table 1 shows the activities of clinical marker enzymes viz. serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, alkaline phosphatase, lactate dehydrogenases and gamma- glu-tamyltransferase, in serum of control and experimental rats. In morphine-induced rats, the level of marker enzymes viz., SGOT (p<0.001), SGPT (p<0.001), ALP (p<0.001), LDH (p<0.01), and [gamma]-GT (p<0.001) were significantly increased in serum when compared with control rats. Whereas in BME pretreated group, these marker enzymes were maintained at near normal level. BME alone treated rats did not show any significant changes in their marker enzymes level when compared with control.
Table 1. Effect of BME on SGOT, SGPT, ALP, LDH and gamma-GT of
control and experimental rats.

Biochemical parameter    Group-I (control)    Group-II (morphine)

SGOT (IU/L)             32.55[+ or -]2.37    41.15[+ or -]1.70***

SGPT (IU/L)             13.03[+ or -]0.66    19.13[+ or -]0.79***

ALP (IU/L)              73.32[+ or -]2.22    81.60[+ or -]1.24***

LDH (IU/L)             314.10[+ or -]20.67  376.82[+ or -]19.48**

[gamma]-GT (IU/L)       9.87[+ or -]0.47     12.52[+ or -]0.60***

Biochemical parameter     Group-III (BME + MR)       Group-IV (BME)

SGOT (IU/L)              31.11[+ or -]2.02 ***    30.5[+ or -] 1.41

SGPT (IU/L)              13.91[+ or -]0.73 ***    13.06[+ or -]0.17

ALP (IU/L)               70.76[+ or -]6.50 **     70.23[+ or -]3.27

LDH (IU/L)             313.67[+ or -]18.81 **   311.93[+ or -]19.83

[gamma]-GT (IU/L)        10.36[+ or -]0.36 ***    10.18[+ or -]0.83

Values are expressed as mean [+ or -] SD, n = 6.
Statistical comparisons are made detween Group II VS. I and Group
III VS. II.
** p-value <0.01.
*** p-value <0.001.


Table 2 shows the level of blood urea, serum creatinine and uric acid in normal and experimental rats. The level of urea (p<0.001), creatinine (p<0.01) and uric acid p<0.001) were found to be significantly higher in the morphine treated group compared to the control group. However, the level of urea (p<0.001), creatinine (p<0.01) and uric acid (p<0.001) were found to be significantly reduced to near normal in BME pretreated rats when compared to morphine-induced rats. BME alone administered rats resembled control rats.
Table 2. Effect of BME on urea, creatinine and uric acid levels
of control and experimental rats.

Groups                    Urea (mg/dl)           Creatinine (mg/dl)

Group I (control)         30.53[+ or -]3.45      2.71[+ or -]0.33

Group I (morphine)        48.68[+ or -]5.47 ***  4.41[+ or -]0.81**

Group I (BME + morphine)  31.07[+ or -]3.59 ***  2.29[+ or -]0.61**

Group IV (BME)            30.13[+ or -]3.27      2.48[+ or -]0.76

Groups                    Uric acid (mg/dl)

Group I (control)         3.76[+ or -]0.69

Group I (morphine)        6.33[+ or -]0.41 ***

Group I (BME + morphine)  3.27[+ or -]0.37 ***

Group IV (BME)            3.23[+ or -]0.57

Values are expressed as mean [+ or -] SD, n = 6.
Statistical comparisons are made between Group II VS. I and
Group III VS. II.
** p-value <0.01.
*** p-value <0.001.


Histopathological examination

Histopathological changes in the liver and kidney of control and experimental rats are shown in Figs. 1 and 2.

Fig. 1 shows the histopathological changes in the liver of control and experimental rats. Fig. 1A illustrates the liver sections of control rats, showing normal hepatic architecture. Fig. 1B shows the architecture of the liver of the rats intoxicated with morphine. The pathological changes observed in morphine intoxicated rats are focal vacuolar degenerative changes (fatty changes) with mononuclear cell infiltration in the hepatocytes. The liver sections of rats pretreated with BME showed recovery of liver tissue from absence of fatty changes and mononuclear cell infiltration in the hepatocytes thereby showing normal hepatic architecture (Fig. 1C). Liver sections of rats given BME alone showed a normal architecture (Fig. 1D).

[FIGURE 1 OMITTED]

Fig. 2 shows the histopathological changes in the kidney of control and experimental rats. Fig. 2A shows the kidney sections of control rats, showing normal architecture. Morphine intoxicated rats, showed mild tubular epithelial cell degeneration with cellular casts within the lumen of the tubules (Fig. 2B). The kidney sections of rats pretreated with BME showed a normal architecture of the kidney, establishing its protective effect against morphine toxicity (Fig. 2C). Kidney sections of rats administered with BME alone did not show any changes in the architecture of the kidney (Fig. 2D).

[FIGURE 2 OMITTED]

HPLC-chromatogram of Bacopa monnieva extract

Fig. 3 shows the overlaid chromatogram of Bacopa monniera extract by HPLC along with Bacosides and the other flavonoids (luteolin and apigenin).

[FIGURE 3 OMITTED]

Discussion

Although opioids are being widely used since very long time, their long-term effects especially at cellular level, are not clearly understood. The liver and kidneys are responsible for the metabolism and excretion of morphine (Coughtrie et al. 1989; Milne et al. 1997). Chronic administration of morphine caused significant elevation in the marker enzyme level in serum. Morphine administration resulted in elevated serum glutamate oxaloacete transaminase and serum glutamate pyruvate transaminase activities. The activity of SGPT increased by 24% and SGOT by 40% after morphine administration (Lurie et al. 1995). A significant increase in the levels of ALT and LDH was reported among chronic heroin users (Panchenko et al. 1999). The increased activities of marker enzyme in serum may be due to leakage of these enzymes from liver as a result of cell damage. Increase in serum transaminases is a common finding in a liver damage. Chronic morphine treatment also caused significant increase in the level of alkaline phosphatase in the serum. In our study we found that the administration of morphine for 21 days caused an increase in alkaline phosphatase activity. The observed elevation in alkaline phosphatase was probably due to the increase in bile pressure commonly seen upon morphine administration. Borzelleca et al. (1994) reported that increased levels of ALT, AST and LDH in rats seen after long-term usage of morphine like agent levo-alphaacetylmethadol(-LAAM) HC1. Chronic administration of morphine also cause an increased levels of urea, uric acid and creatinine in serum. The increased levels of these end products of nitrogen metabolism might be due to the damage caused by long-term effect of morphine in the kidney. In our study continuous administration of morphine, caused a significant increase in creatinine concentration, when compared to control rats. Morphine may cause hepatotoxicity and nephrotoxicity during its metabolism (Van-derlann et al. 1995). Nagamatsu et al. (1986) demonstrated that addition of morphine to the isolated rat hepatocytes induced a marked decrease in the cells and resulted in cell death.

Long-term administration of morphine caused histological alterations in liver and kidney. Histological changes observed in the liver were focal hepatocellular necrosis with mononuclear cell infiltration, mild diffused focal vacuolar degenerative changes in the hepatocytes. Histopathologicaf examination revealed a decrease in vacuolization of the hepatocytes diffusely in the liver, which is indicative of less glycogen storage (Vanderlann et al. 1995). Also our study demonstrates that chronic administration with morphine caused tubular epithelial cell degeneration with cellular casts within the lumen of the tubules in the kidney.

Pretreatment with BME decreases the elevated marker enzyme levels to near normal and this suggested, that the production of structural integrity of hepatocyte cell membrane or regeneration of damaged liver cell by the BME. This indicates the effectiveness of the extract in maintaining the normal function of the liver.

BME pretreated rats did not exhibit any histopathological variations in both liver and kidney, thereby establishing its non-toxic nature on both liver and kidney.

In conclusion, the results of this study showed that BME exerted a protective effect against morphine-induced liver and kidney toxicity. Hence, it merits further development for exploitation as a therapeutic agent. At this stage, however, we do not know the exact mechanism/s responsible for these effects. Further studies are needed to clarify these issues.

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T. Sumathi (a), * S. Niranjali Devaraj (b)

(a) Department of Medical Biochemistry, Dr. A.L.M. Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600113, Tamil Nadu, India

(b) Department of Biochemistry and Bioinformatics, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu, India

* Corresponding author. Tel.: +914424547086; fax: +914424540709.

E-mail address: sumathi_doctor@yahoo.co.in (T. Sumathi).

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doi:10.1016/j.phymed.2009.03.005
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Author:Sumathi, T.; Devaraj, S. Niranjali
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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