Experimental studies on the Renal Protective effect of Gokshura (Tribulus terrestris Linn) and Varuna (Crataeva nurvala Buch-Ham)

Dr. Sudhanshu Kumar Meher¹, Dr. P.K. Mukherjee², Dr. S.K. Banarjee Chaudhury³, Dr. Bani Marjit⁴, Dr. B.P. Shaw⁵

¹Research Officer (Ayurveda), Central Ayurveda Research Institute for Hepatobilliary Disorders, Bhubaneswar.

²Ex-Project Officer, Institute of PG Ayurvedic Education and Research, Kolkata.

³Ex-Professor, Department of Pharmacology, Calcutta National Medical College, Kolkata.

⁴Ex-Professor, Department of Anatomy, Institute of PG Medical Education and Research, Kolkata.

⁵Ex-Professor, Department of Kayachikitsa, IPGAER, Kolkata.

*Corresponding Author E-mail: drmeher@rediffmail.com

ABSTRACT:

Kidney is one of the most vital organs in the body. It is a very sophisticated structure and very complex physiology hence suffers from a variety of specific types of diseases and is also liable to be affected in a number of other systemic conditions of external origin. Moreover, there is no satisfactory treatment for most of the diseases of the kidney, while on the other hand the hospital statistics indicated a rising incidence of kidney diseases in different parts of the world. Standard medical treatment, dialysis and transplant are very expensive propositions and out of reach of the majority hence non-dialytic/non-transplant and economical conservative management is often sought by majority of the patients. In this paper renal protective study has been carried out on aqueous extract of Gokshura (Tribulus terrestris Linn) and Varuna (Crataeva nurvala Buch-Ham) as described in Ayurvedic literature based on their action on urinary system as pre and post treatment in different doses in experimentally induced nephrotoxic animal model with Gentamicin. Parameters like Blood urea and Serum Creatinine estimation, Urine RE/ME and histopathological examination of kidney tissues have been considered for comparative study.. The result has been statistically analyzed for its significance. It is observed that Aqueous Extract of Gokshura Fruit and Aqueous Extract of Varun Bark pre treated to induction of nephrotoxicity shown protection to kidney against Gentamicin induced nephrotoxicity in rats considering the above parameters. The role of these 2 drugs may be considered as renal protective effect against drug induced nephropathy.

KEYWORDS: Renal Protective effect, Gokshura (Tribulus terrestris Linn), Varuna (Crataeva nurvala Buch-Ham), Gentamicin nephrotoxicity.

1. INTRODUCTION:

Kidney is one of the most vital organs in the body. It is a very sophisticated structure and very complex physiology set a unique example of organ differentiation. Similarly, the kidney suffers from a variety of specific types of diseases and is also liable to be affected in a number of other systemic conditions of external origin. Moreover, there is no satisfactory treatment for most of the diseases of the kidney, while on the other hand the hospital statistics indicated a rising incidence of kidney diseases in different parts of the world. All these facts emphasizes the importance of kidney diseases these days and the need for their study in a newer perspective. Again the financial background of the patients of renal problems in India severely limits the choice of treatment. Standard medical treatment, dialysis and transplant are very expensive propositions and out of reach of the majority hence non-dialytic/non-transplant and economical conservative management is often sought by majority of the patients. Looking to this need, renal protective study has been started since few years by experimenting on animals. Various medicinal plants have been described in Ayurvedic literature acting on urinary system. Although no specific description of any drug is found in relation to renal protective effect, their multidimensional actions insist to search the required effect, if any. Experimental research work is a basic and important part of research in medical science to be done in lower animals before initiating any clinical trials on human beings. In this study aqueous extract of Gokshura (Tribulus terrestris Linn) fruit and Varuna (Crataeva nurvala Buch-Ham) stem bark have been used as pre and post treatment in different doses in experimentally induced nephrotoxic animal model with Gentamicin as it is a very commonly used amino glycoside antibiotic having known nephrotoxic potential due to its preferential accumulation in the renal cortical proximal convoluted tubules¹. The trial drugs are well known in the Ayurvedic system of medicine for their beneficial actions on the kidneys like treating urinary tract infection, urolithiasis, burning micturation and various renal disorders².

2. MATERIALS AND METHODS:

2.1 Trial drugs:

The dried fruits of Gokshura (Tribulus terrestris Linn) and stem bark of Varuna (Crataeva nurvala Buch-Ham) were collected as per pharmacopoeial standards. Aqueous extract of both the drugs were prepared with distillation and suction apparatus. Aqueous Extract of Gokshura Fruit (AEGF) was obtained in solid form (5%), grayish in colour and extremely hygroscopic (Photograph 1). Similarly, Aqueous Extract of Varun Bark (AEVB) was obtained in solid form (3%), deep brown in colour and moderately hygroscopic (Photograph 2).

2.2 Determination of LD₅₀ and fixation of dosage of trial drugs:

Graphic method of Miller and Tainter (1944) was adopted for determination of LD₅₀. 20 albino mice inbreed stain average weight 30 gm of either sex were taken in 5 groups for estimation of LD₅₀ofeach of AEGF and AEVB and found to be 325 mg and 350 mg respectively by intra peritoneal route. 2 dosage namely lower and higher dosages of each of trial drugs were evaluated after calculating 20% and 40% of LD₅₀ respectively_.{AEGF - lower dose (65 mg/kg bw), AEGF -higher dose (130 mg/kg bw), AEVB -lower dose (70 mg/kg bw) and AEVB-higher dose (140 mg/kg bw)} for study of renal protective effect.

2.3 Animals:

108 Adult Wistar albino male rats 8 to 10 weeks of age weighting 200gm ± 10 gm were randomly distributed into 18 different groups. As the work had to be done in 3 phases in different time period, 36 animals in each phase under 6 groups had been utilized. The rats were housed in groups of six in metabolic cages at room temperature and with a 12:12 hr light/dark cycle. Animals were provided with commercial food pellets and tap water ad libitum. The different groups were named as group 1 to 18. The drugs were administered orally along with distilled water with the help of curve feeding needle and aseptic 1 ml and 2 ml graduated glass syringes.

Group 1: Served as control - normal saline with food throughout the study without any medication

Group 2: Served as toxicant - Inj. Gentamicin 80 mg/kg bw, intra peritoneal for 8 days

Group 3: Drug treated only AEGF - lower dose (65 mg/kg bw)

Group 4: Drug treated only AEGF - higher dose (130 mg/kg bw)

Group 5: Drug treated only AEVB - lower dose (70 mg/kg bw)

Group 6: Drug treated only AEVB - higher dose (140 mg/kg bw)

Group 7: Pre treated AEGF in toxicant - lower dose (65 mg/kg)

Group 8: Pre treated AEGF in toxicant- higher dose (130 mg/kg)

Group 9: Pre treated AEVB in toxicant - lower dose (70 mg/kg)

Group10: Pre treated AEVB in toxicant-higher dose (140 mg/kg)

Group 11: Pre treated AEGF+ AEVB in toxicant - lower dose (65 mg/kg bw + 70 mg/kg bw)

Group 12: Pre treated AEGF+ AEVB in toxicant - higher dose (130 mg/kg bw + 140 mg/kg bw)

Group 13: Post treated AEGF in toxicant- lower dose (65 mg/kg)

Group 14: Post treated AEGF in toxicant-higher dose (130 mg/kg)

Group 15: Post treated AEVB in toxicant-lower dose (70 mg/kg)

Group 16: Post treated AEVB in toxicant-higher dose (140 mg/kg)

Group 17: Post treated AEGF+ AEVB in toxicant - lower dose (65 mg/kg bw + 70 mg/kg bw)

Group 18: Post treated AEGF+ AEVB in toxicant - higher dose (130 mg/kg bw + 140 mg/kg bw)

2.4 Induction of nephrotoxicity:

Gentamicin was considered as the drug for induction of nephrotoxic model in rats. Inj. Gentamicin 80mg/kg body wt. intraperitoneally for 8 days induced nephrotoxic model³.

2.5 Treatment with trial drugs:

The total study was divided into 3 phases.

Phase 1: This phase aims to evaluate the normal parameters in Group 1 animals, toxicant parameters in group 2 animals and any changes in normal parameters due to trial drugs in the animals of group 3 to 6. On day 12^th and then after every week i.e. on 19^th, 26^th, 33^rd, 40^th and 47^th day urine of one rat from each group were collected for routine (albumin, sugar) and microscopic (casts, cells etc) examination. Metabolic cages were used for collection of urine. Blood sample from those animals were collected by carotid artery puncture under ether anesthesia for estimation of blood urea and serum creatinine with SPAN diagnostic kits. Both kidneys were collected weekly from each rat after sacrifice and light microscopy histological study were conducted.

Phase 2: (Pre treatment phase or preventive phase i.e. trial drug was provided to the animals from 3 days prior to induction of Gentamicin with rats of group 7 to 12). This phase aims to evaluate the effect of pre treatment of the drugs prior to induction of toxicity. 36 rats were maintained as per previously told procedure in 6 groups. Trial drugs were given for 3 days prior to initiating Inj. Gentamicin and during the 8 days of Inj. Gentamicin till collection of samples. On day 12^th and then after every week i.e. on 19^th, 26^th, 33^rd, 40^th and 47^th day urine examination, estimation of blood urea and serum creatinine and histological study of kidneys were conducted.

Phase 3: (Post treatment phase or curative phase i.e. trial drug was provided to the animals after induction of Gentamicin with rats of group 13 to 18). This phase aims to evaluate the effect of post treatment of the drugs after induction of toxicity. 36 rats were maintained as per previously told procedure in 6 groups. Trial drugs were given after initiating Inj. Gentamicin till collection of sample. On day 12^th and then after every week i.e. 19^th, 26^th, 33^rd, 40^th and 47^th day urine examination, estimation of blood urea and serum creatinine and histological study of kidneys were conducted.

2.6 Evaluation of parameters:

2.6.1 Biochemical assays:

Blood urea by DAM (diacetyl monoxime) method and Serum Creatinine by Alkaline Picreate (picric acid) method done calorimetrically were considered as indicator of pathological conditions of kidneys.

2.6.2 Urine examination:

The following factors of urine were observed viz, routine tests like albumin (heat test), sugar (Benedict’s test) and microscopically casts, RBCs, epithelial cells etc. Total urine was centrifuged at 1500 rpm for 15 minutes. Supernatant was used for sugar and albumin estimations and the deposits were emulsified by shaking for microscopic examination. Presence of albumin and sugar was considered to be pathological condition of kidneys. Large, nucleated and usually squamous epithelial cells in large numbers are usually found in toxicant urine as destruction of proximal tubular epithelial cells takes place. Rarely RBCs are found which have no granules so can be distinguished from pus cell⁴.

2.6.3 Histopathological examination:

The histological slides were observed under high power light microscope by paraffin embedding method by following the procedures like tissue fixation, dehydration and clearing, paraffin embedding and block making, section cutting, staining and mounting⁵. The following criteria adopted for grading proximal tubular injury as per the criteria of Houghton et al.⁶ as given below:

Grade 0: Normal

Grade 1: Desquamation of tubular epithelial cells in small foci (less than 1% of total tubule population involved). Areas of focal granulovascular epithelial cell degeneration and granular debris in tubular lumina with or without evidence of desquamation.

Grade 2: Tubular epithelial necrosis and desquamation are prominent but involve less than half of cortical tubules.

Grade 3: More than half of proximal tubules are undergoing necrosis and desquamation, but intact tubules are easily identified.

Grade 4: Total or near total proximal tubular necrosis.

2.7 Statistical analysis:

This was carried out with Fisher ‘t’ or Student ‘t’ test as the sample size is very small and comparison was made between two measurements in 2 different groups i.e. for testing the significance between independent sample means. Probability value less than 0.1 was considered significant.

3. RESULTS AND DISCUSSION:

3.1 Blood Urea:

The mean serum urea (SU) of control rats is estimated to be 36.75 mg/dl. The SU in the rats of group 2 (toxicant) showed a high increase initially and gradually decrease. The statistical analysis showed a highly significant rise (P<0.001) in comparison to group 1 rats. It was gradually decreasing in weekly study. In other groups of phase 1 study viz, in group 3, 4, 5 and 6, the difference of SU is insignificant (P>0.5) in comparison to control group. It revealed that AEGF and AEVB do not alter the SU if treated in lower as well as higher doses (Table 1). The pretreatment phase study showed that there was highly significant reduction of SU in group 12, pre treated with AEGF 130 mg/kg bw + AEVB 140 mg/kg bw in comparison to toxicant i.e. group 2 (P<0.01). The effects of other groups have been depicted in the table (Table 2). The post treatment phase study showed that there is moderate significant reduction of SU in group 18 post treated with AEGF 130 mg/kg bw + AEVB 140 mg/kg bw in comparison to toxicant i.e. group 2 (P<0.1). Even after full course of treatment SU did not came to normal. The effects of other groups have been depicted in the table (Table 3). It is observed from table 2 and 3 that pretreated group with higher doses of AEGF and AEVB (group 12) showed more significant reduction of SU as compared to post treatment group with higher doses of AEGF and AEVB (group 18). The effects of other groups have been depicted in the table.

Table 1: Showing the Serum Urea (mg/dl) levels of rats of phase 1 study (Group 1 to Group 6)

Day	Group 1⁺ Control	Group 2 Toxicant Inj. Gentamicin 80mg/kg bw i.p. X 8 days	Group 3 AEGF treated 65mg/kg	Group 4 AEGF treated 130mg/kg	Group 5 AEVB treated 70mg/kg	Group 6 AEVB treated 140mg/kg
12^th	39.0	180	40	39	40	39
19^th	37.5	165	38	37	39	37
26^th	36.0	150	36	35	36	35
33^rd	34.5	135	34	35	37	37
40^th	36.0	117	37	37	36	37
47^th	37.5	81	36	36	34	36
Fister ‘t’		6.95	0.076	0.280	0.227	0.095
P value		< 0.001*	> 0.5*	> 0.5*	> 0.5*	> 0.5*

⁺ SU Mean ±SE = 36.75±0.64 * Compared with Group 1

Table 2: Showing the Serum Urea (mg/dl) levels of rats of phase 2 study (Group 7 to Group 12) -Pre treatment phase

Day	Group 7 AEGF 65mg/kg pre treated	Group 8 AEGF 130mg/kg pre treated	Group 9 AEVB 70mg/kg pre treated	Group 10 AEVB 140mg/kg pre treated	Group 11 AEGF 65 mg/kg + AEVB 70 mg/kg pre treated	Group 12 AEGF 130 mg/kg + AEVB 140 mg/kg pre treated
12^th	165	140	172	150	124	105
19^th	154	135	165	145	120	95
26^th	148	130	147	140	115	75
33^rd	135	122	135	130	107	39
40^th	115	115	117	110	100	39
47^th	75	68	73	75	58	39
Fister ‘t’	0.30	1.08	0.152	0.70043	1.93	3.80
P value	> 0.5	< 0.05*	> 0.5*	< 0.5*	< 0.1*	< 0.01*

* Compared with Group 2

Table 3: Showing the Serum Urea (mg/dl) levels of rats of phase 3 study (Group 13 to Group 18) -Post treatment phase

Day	Group 13AEGF 65mg/kg post treated	Group 14 AEGF 130mg/kg post treated	Group 15 AEVB 70mg/kg post treated	Group 16 AEVB 140mg/kg post treated	Group 17 AEGF 65 mg/kg + AEVB 70 mg/kg post treated	Group 18 AEGF 130 mg/kg + AEVB 140 mg/kg post treated
12^th	172	150	175	160	140	120
19^th	157	145	167	155	136	115
26^th	150	140	149	150	130	109
33^rd	135	130	135	135	120	103
40^th	117	110	117	110	115	97
47^th	78	75	75	72	71	55
Fister ‘t’	0.158	0.70043	0.080	0.38	1.086	2.19
P value	> 0.5*	< 0.5*	> 0.5*	> 0.5*	< 0.5*	< 0.1*

* Compared with Group 2

Table 4: Showing the Serum Creatinine (mg/dl) levels of rats of phase 1 study (Group 1 to Group 6)

Day	Group 1^# Control	Group 2 Toxicant Inj. Gentamicin 80mg/kg bw i.p. X 8 days	Group 3 AEGF treated 65mg/kg	Group 4 AEGF treated 130mg/kg	Group 5 AEVB treated 70mg/kg	Group 6 AEVB treated 140mg/kg
12^th	0.42	2.35	0.42	0.31	0.31	0.21
19^th	0.21	1.92	0.31	0.21	0.31	0.42
26^th	0.31	1.74	0.21	0.31	0.21	0.31
33^rd	0.21	1.28	0.31	0.42	0.31	0.31
40^th	0.31	0.85	0.21	0.21	0.42	0.21
47^th	0.42	0.64	0.42	0.31	0.21	0.21
Fister ‘t’		4.25	0	0.373	0.373	0.678
P value		< 0.01*	> 0.5*	> 0.5*	> 0.5*	> 0.5*

^# SC Mean ±SE = 0.3133±0.038 * Compared with Group 1

Table 5: Showing the Serum Creatinine (mg/dl) levels of rats of phase 2 study (Group 7 to Group 12)-Pre treatment phase

Day	Group 7 AEGF 65mg/kg pre treated	Group 8 AEGF 130mg/kg pre treated	Group 9 AEVB 70mg/kg pre treated	Group 10 AEVB 140mg/kg pre treated	Group 11 AEGF 65 mg/kg + AEVB 70 mg/kg pre treated	Group 12 AEGF 130 mg/kg + AEVB 140 mg/kg pre treated
12^th	1.85	1.78	1.91	1.90	1.75	1.50
19^th	1.65	1.62	1.79	1.75	1.50	1.28
26^th	1.60	1.35	1.65	1.50	1.08	0.85
33^rd	1.15	1.20	1.20	1.10	0.90	0.42
40^th	0.80	0.76	0.85	0.80	0.64	0.31
47^th	0.60	0.46	0.60	0.60	0.40	0.31
Fister ‘t’	0.547	0.784	0.376	0.539	1.22	2.0
P value	> 0.5	< 0.5*	> 0.5*	< 0.5*	< 0.1*	< 0.1*

* Compared with Group 2

Table 6: Showing the Serum Creatinine (mg/dl) levels of rats of phase 3 study (Group 13 to Group 18) -Post treatment phase

Day	Group 13 AEGF 65mg/kg post treated	Group 14 AEGF 130mg/kg post treated	Group 15 AEVB 70mg/kg post treated	Group 16 AEVB 140mg/kg post treated	Group 17 AEGF 65 mg/kg + AEVB 70 mg/kg post treated	Group 18 AEGF 130 mg/kg + AEVB 140 mg/kg post treated
12^th	1.90	1.80	1.95	1.85	1.80	1.74
19^th	1.80	1.60	1.83	1.65	1.60	1.50
26^th	1.65	1.54	1.69	1.60	1.34	1.07
33^rd	1.20	1.08	1.24	1.15	1.18	0.85
40^th	0.85	0.80	0.85	0.80	0.75	0.64
47^th	0.60	0.60	0.60	0.60	0.45	0.42
Fister ‘t’	0.376	0.692	0.285	0.547	0.825	1.255
P value	> 0.5*	> 0.5*	> 0.5*	> 0.5*	> 0.5*	< 0.5*

* Compared with Group 2

Table 7: Showing presence of Urinary Constituents of rats Group 1 to Group 6

Day	Group 1	Group 2	Group 3	Group 4	Group 5	Group 6
12th	-	A,S,HC,GC,EC,RBC	-	-	-	-
19th	-	A,S,HC,GC,EC	-	-	-	-
26th	-	S,HC,GC	-	-	-	-
33rd	-	S,GC	-	-	-	-
40th	-	-	-	-	-	-
47th	-	-	-	-	-	-

Table 8: Showing presence of Urinary Constituents of rats Group 7 to Group 12

Day	Group 7	Group 8	Group 9	Group 10	Group 11	Group 12
12th	S,GC,RBC	S,GC	S,GC,RBC	S,GC	EC,GC	EC
19th	S,GC	S	S,GC	S	EC	-
26th	GC	-	GC	-	-	-
33rd	-	-	-	-	-	-
40th	-	-	-	-	-	-
47th	-	-	-	-	-	-

Table 9: Showing presence of Urinary Constituents of rats Group 13 to Group 18

Day	Group 13	Group 14	Group 15	Group 16	Group 17	Group 18
12^th	A,S,HC,GC,EC,RBC	A,S,HC,GC,EC	A,S,HC,GC,EC,RBC	A,S,HC,GC	S,HC,GC	HC, S,EC
19^th	A,S,HC,GC,EC	S,GC	A,S,HC,GC,EC	S,GC	HC,EC	HC
26^th	S,HC,GC,EC	GC	S,HC,GC,EC	GC	EC	HC
33^rd	HC,EC	-	HC,EC	-	-	-
40^th	-	-	-	-	-	-
47^th	-	-	-	-	-	-

Table 10: Grading of histopathological picture of rat kidneys of Group 1 to Group 6

Day	Group 1	Group 2	Group 3	Group 4	Group 5	Group 6
12th	0	4	0	0	0	0
19th	0	4	0	0	0	0
26th	0	4	0	0	0	0
33rd	0	3	0	0	0	0
40th	0	3	0	0	0	0
47th	0	2	0	0	0	0

Table 11: Grading of histopathological picture of rat kidneys of Group 7 to Group 12

Day	Group 7	Group 8	Group 9	Group 10	Group 11	Group 12
12th	4	3	4	3	3	3
19th	3	3	3	3	3	3
26th	3	2	3	3	2	2
33rd	3	2	3	2	2	1
40th	2	1	2	2	1	1
47th	2	1	2	1	1	0

Table 12: Grading of histopathological picture of rat kidneys of Group 13 to Group 18

Day	Group 13	Group 14	Group 15	Group 16	Group 17	Group 18
12^th	4	3	4	3	3	3
19^th	4	3	4	3	3	3
26^th	3	3	3	3	3	2
33^rd	3	3	3	3	2	2
40^th	3	2	3	2	2	2
47^th	2	2	2	2	1	1