Journul of Elhnopharmacology, Elsevier Scientific Publishers
EFFECT OF CRATAEVA UROLITHIASIS
P. VARALAKSHMI,
Department (Accepted
Y. SHAMILA
NURVALA
IN EXPERIMENTAL
and E. LATHA
of Medical Biochemistry, Postgraduate Madras-600 119 llndti
University,
313
28 (1990) 313- 321 Ireland Ltd.
Institute
of Basic Medical Sciences, Madras
Taramani
October 10, 1989)
Summary
The effect of oral administration of Crataeva nurvalu bark deco&ion on calcium oxalate lithiasis has been studied in rats. The elevation of the oxalate-synthesizing liver enzyme, glycolate oxidase, produced by feeding glycollit acid was remarkably reduced with the decoction, showing a regulatory action on endogenous oxalate synthesis. Protein-bound carbohydrates were increased in the renal tissues during calculosis but these changes were not reversed with the herbal treatment. The increased deposition of stone-forming constituents in the kidneys of calculogenic rats was lowered with decoction administration. The increased urinary excretion of the crystalline constituents along with lowered magnesium excretion found in stone-forming rats was partially reversed by decoction treatment.
Introduction Crataeva nurva2.a Buch religiosa Hook et Forst.)
Ham (family Capparidaceae; synonmy Crataeva is one of the medicinal plants in the Indian Ayurvedic system of medicine. The root and the bark are reported to have antilithic actions (Nadkarni, 1954; Kumar et al., 1980). It is also mentioned in the old samhitas Charak (210 B.C.- 170 A.D.) and Sushrata (176 A.D.-340 A.D.) for the treatment of urinary calculi. It is commonly known in English as Capparis trifoliata (three-leaved caper), as Pashuganda or Asmarigha in Sanskrit and as Varuna in Hindi. It has a wide distribution in India. Earlier work from this laboratory has shown a male preponderance of kidney stones with most patients harbouring calcium oxalate in their stones Waralakshmi et al., 1976). Individuals were also recurrent stone formers, exhibiting mild hyperoxaluria (Varalakshmi and Anandam, 1979) and were difficult to treat effectively. In the present paper, preliminary investigations have been carried out in experimental calcium oxalate stone-forming male rats, with a view to study the role of this herbal drug in stone disorders. 0378-8741/$03.50 0 1990 Elsevier Published and Printed in Ireland
Scientific Publishers
Ireland Ltd.
314
Materials and methods Preparation
of bark decoction
C. nurvalu bark was obtained locally from an Ayurvedic medical shop and the identity confirmed by the Chief Botanist at the Indigenous Medical Hospital. The decoction was prepared, generally following the procedure of Nadkarni (19541 by bruising and boiling 120 g of powdered bark in 750 ml of distilled water, then straining through muslin and cooling. This final product was used directly for experimental purposes. Experimental
animals
Inbred male albino rats of the Wistar strain (150- 180 g) were fed standard pelleted diet (Gold Mohur, Hindustan Lever Ltd., India) and water was given ad libitum. They were acclimatized to the animal house conditions for a week. They were then divided into four groups comprising six animals in each. Group I received the commercial diet and served as controls. Group II was administered a calculi-producing diet (CPD: commercial diet mixed with 3% glycollic acid) for 40 days (Chow et al., 19741. Group III received the commercial diet for 40 days with the decoction being administered during the last 10 days. Group IV was fed CPD for the same duration, with the decoction treatment given for the last 10 days. The bark decoction was administered orally by stomach tube (2 ml/rat/day). This amount is equivalent to 80 mg of the starting powder. The initial and final body weights were recorded at the start of the experimental period and just before being killed. Collection and analysis of urine samples
On the day before being killed, the rats were housed in metabolic cages for 24-h urine collections. A drop of concentrated HCl was added to the urine before being stored at 4OC. Oxalate, calcium, inorganic phosphorus and magnesium contents were determined using the respective procedures of Hodgkinson and Williams (19721, Hooper (19561, Fiske and Subbarow (1925) and Neil1 and Neely (19561. Assay
Of tiSSUQQTLZymQ
At the end of the experimental period, the animals were killed by decapitation and livers and kidneys excised, washed with cold 0.15 M KC1 and weights recorded. A 10% homogenate of the tissues were prepared in 0.01 M phosphate buffer (pH 7.0) and centrifuged at 12,000 x g for 30 min. The supernatant was used for the enzyme assays. Two major oxalate-synthesizing enzymes, namely, glycolate oxidase (GAO) and lactate dehydrogenase (LDH) were assayed in the liver (Murthy et al., 1981; King, 1965). In addition, inorganic pyrophosphatase (PPasel and the acid and alkaline phosphatases (ACP and ALP) were also determined (Jesse, 1966; King, 1965). Protein estimation was performed in all tissues by the method of Lowry et al. (19511.
315
Kidney homogenates were also subjected to all of the above assays for GAO, since that tissue is devoid of the enzyme.
except
Renal tissue deposition of stone constituents Wet ashing of kidney tissue was done according to the method of Ballentine and Barford (19571 and used for estimating calcium, phosphorus and oxalate in order to assess the extent of deposition of crystalline components. Estimation of protein-bound carbohydrates in the kidney Defatted kidney tissue after hydrolysis was used for estimating the protein-bound carbohydrates hexosamine, sialic acid and hexuronic acid (Wagner, 1979; Niebes, 1972; (Bitter and Muir, 19621. Results and discussion
Glycolate has been used successfully as an inducer of oxalate lithiasis in rats (Chow et al., 19741. Liver is the major site of endogenous oxalate synthesis. The conversion of glycolate to oxalate is catalysed by the liver enzyme GAO, through glyoxylate as the intermediate (Richardson and Tolbert, 1961). It is a flavin-linked peroxisomal enzyme, localised predominantly in the liver (Masters and Holmes, 1977). The other major enzyme, LDH, has a ubiquitous distribution and is cytosolic. It can convert glyoxylate to oxalate as well as to glycolate by simultaneous oxidation and reduction (Williams and Smith, 19711. The greater significance of liver GAO in endogenous oxalate synthesis over that of LDH has been established (Varalakshmi and Richardson, 1983a). Effect of the liver enzymes Increased GAO activity was observed in the livers of rats receiving CPD (Table 1, Group II) relative to animals on a normal diet (Group I). Administration of the bark deco&ion to such animals produced a considerable lowering enzyme activity (Group III) and the decoction restored the activity to that of normals in the CPD-fed group (Group IV). A similar observation in the enzyme level has been observed in vitamin B6 deficiency (Varalakshmi and Richardson, 1983131.The lowering effect on the enzyme level by the herbal deco&ion assigns it an important role as a regulator of endogenous oxalate synthesis. Liver LDH remained unaltered in the stone-forming rats as well as in the deco&ion-treated groups. ALP was increased in rats receiving calculogenic food (Group II) relative to rats receiving the control diet (Group Il. There was a slight decrease in the PPase activity in the above group. C. nurvala decoction treatment lowered the ACP level in both normals and stone-formers, but PPase was unaffected. The latter enzyme does not take part in extracellular calcification, rather the action of it is only to hydrolyse inorganic pyrophosphate within the cell.
CPD AND C. NiIltVALA
DECGCTIGN
0.43 0.40 1.58 0.33
LDH PPase ACP ALP
0.45 0.31 1.52 0.46
rt f +: f
0.16 o-05”* 0.04 0.04***
4.92 F 0.11*****
Group II
0.43 0.39 1.09 0.32
+ 2 + 2
0.02 0.02 o.o4b.*** 0.07
1.89 I- O.Ogb,***
Group HI
Comparisons were made between groups 4 and II, bI and III, ‘I and IV, W and IV and HI and IV. Statistical ***p < 0.001.
0.02 0.06 0.07 0.08
2.80 + 0.15
GAO
k F k f
Group I
Enzymes
significance:
+ f + f
0.01 o.o3d,* 0.25”,* 0.03da** *P < 0.05; **P < 0.01;
0.44 0.38 1.39 0.36
3.01 f 9 ~~‘.*:d,***:e,4#*
Group IV
Tabular values are mean it S.D. for 6 animals. Enzyme units are expressed for GAO as nmol of glyoxylate/min/mg protein; LDH as pmol of pyruvatelminlmg protein; PPase as pm01 of phosphorushninlmg protein; ACP and ALP as nmol of phenol/min/mg protein, all at 37%.
ENZYMATIC CHANGES IN THE LIVERS OF RATS RECEIVING
TABLE 1
CARBOHYDRATES
0.96 f 0.08”
Comparisons were made between +**p < 0.001.
0.01 0.01 0.25 0.16ba***
0.842 0.07
12.75 rt 0.57 1.142 0.14
+ + + r
Group III
groups ‘1 and II, “1 and III,
EFFECT OF CRATAEVA UROLITHIASIS
P. VARALAKSHMI,
Department (Accepted
Y. SHAMILA
NURVALA
IN EXPERIMENTAL
and E. LATHA
of Medical Biochemistry, Postgraduate Madras-600 119 llndti
University,
313
28 (1990) 313- 321 Ireland Ltd.
Institute
of Basic Medical Sciences, Madras
Taramani
October 10, 1989)
Summary
The effect of oral administration of Crataeva nurvalu bark deco&ion on calcium oxalate lithiasis has been studied in rats. The elevation of the oxalate-synthesizing liver enzyme, glycolate oxidase, produced by feeding glycollit acid was remarkably reduced with the decoction, showing a regulatory action on endogenous oxalate synthesis. Protein-bound carbohydrates were increased in the renal tissues during calculosis but these changes were not reversed with the herbal treatment. The increased deposition of stone-forming constituents in the kidneys of calculogenic rats was lowered with decoction administration. The increased urinary excretion of the crystalline constituents along with lowered magnesium excretion found in stone-forming rats was partially reversed by decoction treatment.
Introduction Crataeva nurva2.a Buch religiosa Hook et Forst.)
Ham (family Capparidaceae; synonmy Crataeva is one of the medicinal plants in the Indian Ayurvedic system of medicine. The root and the bark are reported to have antilithic actions (Nadkarni, 1954; Kumar et al., 1980). It is also mentioned in the old samhitas Charak (210 B.C.- 170 A.D.) and Sushrata (176 A.D.-340 A.D.) for the treatment of urinary calculi. It is commonly known in English as Capparis trifoliata (three-leaved caper), as Pashuganda or Asmarigha in Sanskrit and as Varuna in Hindi. It has a wide distribution in India. Earlier work from this laboratory has shown a male preponderance of kidney stones with most patients harbouring calcium oxalate in their stones Waralakshmi et al., 1976). Individuals were also recurrent stone formers, exhibiting mild hyperoxaluria (Varalakshmi and Anandam, 1979) and were difficult to treat effectively. In the present paper, preliminary investigations have been carried out in experimental calcium oxalate stone-forming male rats, with a view to study the role of this herbal drug in stone disorders. 0378-8741/$03.50 0 1990 Elsevier Published and Printed in Ireland
Scientific Publishers
Ireland Ltd.
314
Materials and methods Preparation
of bark decoction
C. nurvalu bark was obtained locally from an Ayurvedic medical shop and the identity confirmed by the Chief Botanist at the Indigenous Medical Hospital. The decoction was prepared, generally following the procedure of Nadkarni (19541 by bruising and boiling 120 g of powdered bark in 750 ml of distilled water, then straining through muslin and cooling. This final product was used directly for experimental purposes. Experimental
animals
Inbred male albino rats of the Wistar strain (150- 180 g) were fed standard pelleted diet (Gold Mohur, Hindustan Lever Ltd., India) and water was given ad libitum. They were acclimatized to the animal house conditions for a week. They were then divided into four groups comprising six animals in each. Group I received the commercial diet and served as controls. Group II was administered a calculi-producing diet (CPD: commercial diet mixed with 3% glycollic acid) for 40 days (Chow et al., 19741. Group III received the commercial diet for 40 days with the decoction being administered during the last 10 days. Group IV was fed CPD for the same duration, with the decoction treatment given for the last 10 days. The bark decoction was administered orally by stomach tube (2 ml/rat/day). This amount is equivalent to 80 mg of the starting powder. The initial and final body weights were recorded at the start of the experimental period and just before being killed. Collection and analysis of urine samples
On the day before being killed, the rats were housed in metabolic cages for 24-h urine collections. A drop of concentrated HCl was added to the urine before being stored at 4OC. Oxalate, calcium, inorganic phosphorus and magnesium contents were determined using the respective procedures of Hodgkinson and Williams (19721, Hooper (19561, Fiske and Subbarow (1925) and Neil1 and Neely (19561. Assay
Of tiSSUQQTLZymQ
At the end of the experimental period, the animals were killed by decapitation and livers and kidneys excised, washed with cold 0.15 M KC1 and weights recorded. A 10% homogenate of the tissues were prepared in 0.01 M phosphate buffer (pH 7.0) and centrifuged at 12,000 x g for 30 min. The supernatant was used for the enzyme assays. Two major oxalate-synthesizing enzymes, namely, glycolate oxidase (GAO) and lactate dehydrogenase (LDH) were assayed in the liver (Murthy et al., 1981; King, 1965). In addition, inorganic pyrophosphatase (PPasel and the acid and alkaline phosphatases (ACP and ALP) were also determined (Jesse, 1966; King, 1965). Protein estimation was performed in all tissues by the method of Lowry et al. (19511.
315
Kidney homogenates were also subjected to all of the above assays for GAO, since that tissue is devoid of the enzyme.
except
Renal tissue deposition of stone constituents Wet ashing of kidney tissue was done according to the method of Ballentine and Barford (19571 and used for estimating calcium, phosphorus and oxalate in order to assess the extent of deposition of crystalline components. Estimation of protein-bound carbohydrates in the kidney Defatted kidney tissue after hydrolysis was used for estimating the protein-bound carbohydrates hexosamine, sialic acid and hexuronic acid (Wagner, 1979; Niebes, 1972; (Bitter and Muir, 19621. Results and discussion
Glycolate has been used successfully as an inducer of oxalate lithiasis in rats (Chow et al., 19741. Liver is the major site of endogenous oxalate synthesis. The conversion of glycolate to oxalate is catalysed by the liver enzyme GAO, through glyoxylate as the intermediate (Richardson and Tolbert, 1961). It is a flavin-linked peroxisomal enzyme, localised predominantly in the liver (Masters and Holmes, 1977). The other major enzyme, LDH, has a ubiquitous distribution and is cytosolic. It can convert glyoxylate to oxalate as well as to glycolate by simultaneous oxidation and reduction (Williams and Smith, 19711. The greater significance of liver GAO in endogenous oxalate synthesis over that of LDH has been established (Varalakshmi and Richardson, 1983a). Effect of the liver enzymes Increased GAO activity was observed in the livers of rats receiving CPD (Table 1, Group II) relative to animals on a normal diet (Group I). Administration of the bark deco&ion to such animals produced a considerable lowering enzyme activity (Group III) and the decoction restored the activity to that of normals in the CPD-fed group (Group IV). A similar observation in the enzyme level has been observed in vitamin B6 deficiency (Varalakshmi and Richardson, 1983131.The lowering effect on the enzyme level by the herbal deco&ion assigns it an important role as a regulator of endogenous oxalate synthesis. Liver LDH remained unaltered in the stone-forming rats as well as in the deco&ion-treated groups. ALP was increased in rats receiving calculogenic food (Group II) relative to rats receiving the control diet (Group Il. There was a slight decrease in the PPase activity in the above group. C. nurvala decoction treatment lowered the ACP level in both normals and stone-formers, but PPase was unaffected. The latter enzyme does not take part in extracellular calcification, rather the action of it is only to hydrolyse inorganic pyrophosphate within the cell.
CPD AND C. NiIltVALA
DECGCTIGN
0.43 0.40 1.58 0.33
LDH PPase ACP ALP
0.45 0.31 1.52 0.46
rt f +: f
0.16 o-05”* 0.04 0.04***
4.92 F 0.11*****
Group II
0.43 0.39 1.09 0.32
+ 2 + 2
0.02 0.02 o.o4b.*** 0.07
1.89 I- O.Ogb,***
Group HI
Comparisons were made between groups 4 and II, bI and III, ‘I and IV, W and IV and HI and IV. Statistical ***p < 0.001.
0.02 0.06 0.07 0.08
2.80 + 0.15
GAO
k F k f
Group I
Enzymes
significance:
+ f + f
0.01 o.o3d,* 0.25”,* 0.03da** *P < 0.05; **P < 0.01;
0.44 0.38 1.39 0.36
3.01 f 9 ~~‘.*:d,***:e,4#*
Group IV
Tabular values are mean it S.D. for 6 animals. Enzyme units are expressed for GAO as nmol of glyoxylate/min/mg protein; LDH as pmol of pyruvatelminlmg protein; PPase as pm01 of phosphorushninlmg protein; ACP and ALP as nmol of phenol/min/mg protein, all at 37%.
ENZYMATIC CHANGES IN THE LIVERS OF RATS RECEIVING
TABLE 1
CARBOHYDRATES
0.96 f 0.08”
Comparisons were made between +**p < 0.001.
0.01 0.01 0.25 0.16ba***
0.842 0.07
12.75 rt 0.57 1.142 0.14
+ + + r
Group III
groups ‘1 and II, “1 and III,
