Indian Journal of Experimental Biology Vol. 39, October 2001, pp. 1022-1027

Hepatoprotective action of abhrak bhasma, an Ayurvedic drug in albino rats against hepatitis induced by CC14

Savita Buwa, Subhash Patil*, PH Kulkarni* & Aruna Kanase**

Cell Biology Section, Department of Zoology, Shivaji University, Kolhapur 410 006, India *Institute of Indian Medici ne, Kothrud, Pune 411 029, India

Received 20 September 2000; revised 6 June 2001

Abhrak bhasma is a commonly used ayurved ic drug against many diseases including hepatiti s. It is tes ted in albino rats using a model of hepatitis induced by a single dose of CCI4 (3 mllkg body wt). Different doses of abhrak bhasma ( I 0, 20, 30 and 40 mg/kg body wt) were tested to decide the dose re lated hepatoprotective efficacy. The centrolobular necrosis induced by single dose of CCI4 was reduced significantly by abhrak bhasma (10 mg) and liver histology was also protected by 20 mg dose. Liver ac id lipase activity was lowered, while alkaline and lipoprotein lipase ac ti vities were elevated due to treatment of single dose of CCI4. Abhrak bhas ma counteracted the action of CCI4 on li ver lipolytic enzymes. CCI4 did not alter the kidney histologically. Activities of three lipases of rat kidney (acid, a lkaline and lipoprotein lipases) were reduced by CCI4

treatment and were reversed by administration of abhrak bhasma. Acid lipase act ivity of rat adipose ti ssue was reduced by CCI 4 treatment. On the contrary alkaline, lipoprotein and hormone sensi ti ve lipases were enhanced after 24 hr of admini stration of CCI4 . Acid lipase act ivity was raised by administration of different doses of abhrak bhasma concurrent with CCI4. Abhrak bhasma treatment along with CCI4 enhanced alkaline lipase activi ty at 10 and 20 mg dose and later it was reduced at 30 and 40 mg doses and came to normal levels. Lipoprotein and hormone sensitiv'e lipases were reduced by the counteraction of increasing doses of abhrak bhasma.

Lipases play roles in lipid metabolism and lipid turnover. Lipoprotein lipase (LPL) degrades chylomi­crons and free fatty acids are uptaken by the different tissues 1

• Hormone sensitive lipase (HSL) hydrolyses trigl ycerides to monoglycerides for their mobilization from adipose ti ssue to different tissues and cells2

.

Hepatic lysosomal lipase/acid lipase (ACL) ts involved in degradation of lipid material for recirculation of fatty acids3

. Alkaline lipase is supposed to be microsomal and cytosolic and is involved in lipid synthesis4. Our earlier work on lipolytic activities during hepatoprotection mediated by different ayurvedic drugs has shown that lipases are influenced by the status of hepatitis, hepato­protection by ayurvedic drugs and conesponding altered physiology of the animals. Mandur bhasma ( l 0 mglkg body wt; PO) administration to CCl4 + liquid paraffin (CC14 in liquid paraffin 3:1 v/v/kg body wt) treated rats for 11 days res ulted in hepatoprotection through regeneration of liver ev idenced by mitosis coupled with renal protection5

.

Liver lipoprotein lipase was increased en hancing the

**Correspondent author: dlpat il @pn3.vsnl. net.in & arunakanase@ usa. net

secretion of hepatic lipoproteins coupled with lowered acid lipase and hormone sensitive lipase (to indicate less lipolysis than uptake of fatty acids). CC14 induced lowered activities of lysosomal acid lipases from liver and kidney counteracted by mandur bhasma by releasing fatty acids for regeneration with increased activities of alkaline lipases which are also needed for regeneration. Patil et a/. 6 have show n similar alter­ations in activi ti es of acid, lipoprotein and hormone sensitive lipases during kumari asav , kumari kaJpa, arogyavardhini and tamra bhasma mediated differ­ential hepatoprotection when these drugs have been given to the rats simultaneous with CC14 for 7 days . These four ayurvedic drugs when used in the other mode of toxicity, where CC14 has been administered once in a week for 4 weeks alkaline lipase acti vity decreases, but lipoprotein lipase and hormone sensitive lipase activities increase 7 . These differences in the alterations have been attributed to the modes of toxicities. Single dose of CC14 induces acute centro­lobular necrosis and alters the lipid metabolisrn8

. CC14 (0.1 mL/100 g body wt) introduced intraduodenally, increases liver tri- and di-g lycerides levels after 24 hr in albino rats with centrolobular necrosis, which are influenced by dietary lipids9

. But CC14 (0.5 mL/100 g

BUWA et al.: HEPATOPROTECTIVE ACTION OF ABHRAK BHASMA 1023

body wt) elevates the levels of medium C-chain fatty acids (which are decreased by lipid balanced diet) in adipose tissue and not in liver and decreases saturated fatty acids (without any alteration in dietary linoleic acid and linolenic acid) in rat. The repair mechanisms are influenced by phospholipids (phosphotidylcholine being more pronounced in action) 10 and coupled with rise in levels of thyrnidylate synthetase and thymidine kinase in liver with reaching peaks at 72 hr indica ting liver regeneration 11

• Thus lipid metabolism is influenced by both toxicity by CC14 and regeneration of liver.

It has been shown in this laboratory that mandur bhasma, tamra bhasma, arogyavardhini, kuamri asav and kumari kalpa exhibited hepatoprotective activity and caused significant alterations in lipolytic activity of liver, kidney and ad ipose tissue of albino rat5

•7

•

Abhrak bhasma exhibited hepatoprotective potency by lowering serum enzymes and bilirubin against liver injury by single dose of CC14

12. In the ptesent study an

attempt has been made to elucidate the effects of varying doses of abhrak bhasma on lipolytic activities of liver, kidney and adipose tissue during hepatic injury induced by single dose of CCI4•

Materials and Methods Abhrak bhasma was prepared in the laboratory as

described in Rasa ratna Sammuchchaya 13. Albino rats

were bred and reared in Departmental animal house. The rats were originally derived from Haffkine strai n. ThP.y were provided standard pellet feed (Amrit rat feed prepared by Navbharat Chakan Oil Mills, Sangli, India) and were given water ad labium. The rats weighing 130 to 140 g were used for the present experiment. The animals were grouped into 6 groups (each containing 8 animals). The rats of first group were designated as normal and were not given any treatment. The rats of the groups 2 to 6 were given single injection of CC14 (3 mL!kg body wt). The rats of the groups 3 to 6 were also given abhrak bhasma (10, 20, 30 and 40 mg/kg body wt respectively; po) immediately after the injection of CCI4• Animals were deprived of food 12 hr prior to sacrificing. The rats were sacrificed after 24 hr of treatment by giving deep ether anaesthesia. Livers, kidneys and. adipose tissues were dissected out. The pieces of livers and kidneys were fixed in a fixative containing paraformaldehyde ( 4%) and glutaraldehyde (1%) in phosphate buffer (0.2 M; pH 7.00) and processed for the preparation of paraffin blocks. The sections were cut (5 f..tm) and

stained with hematoxylin and eosin and Mallory's triple stain. The tissues were homogenised and diluted to a suitable volume with distilled water and used for the assays of lipases. Acid lipase was determined according to the method of Mahadevan and Tappel3

and alkaline, lipoprotein and hormone sensitive lipases were assayed in tissue homogenates as described earlier by Matsumura et al. 14 using triolein as a substrate. The enzyme activities were stopped at the end of incubation period by addition of 2 mL of Cu-triethanolamine reagent and fatty acids liberated were estimated as described by ltaya 15

. The proteins of livers and kidneys were estimated using Folin phenol reagent16

. Proteins from adipose tissues were estimated using the method described by Tornqvist and Belfrage 17

. Student's t test was carried out according to Agarwai 18

.

Results and Discussion Figure l exhibits normal architecture of group I rat

liver. Single dose of CC14 caused centrolobular necrosis in liver (Fig. 2). Periarterial region remained normal with the treatment of CCI4 (3.0 mllkg body wt). The cells of centrolobular region showed vacuo­lated cytoplasm. Vacuolar size showed variations from small spherical to large droplet like structures. In most of the necrotic cells centrally placed nuclei were suspended in small amcunt of cytoplasm which remains continuous by cytoplasmic strands that traverse through the vacuoles connecting peripheral rim of cytoplasm. Many of them were dead. Kupffer cells and si nusoidal cells showed arrest in distribution . Similar necrosis was described elsewhere and described as hydropic degeneration/ fatty degeneration8

• 19

"21

. Abhrak bhasma (10 mg/kg body wt) given orally along with CCI4 protected the liver partially (Fig. 3). The centrolobular region still showed necrosis, but the extent of the area of necrotic region was reduced significantly . Numbers of necrotic cells located in this region were considerably reduced and were retained in immediate vicinity of the vein. Most of the cells on the boundary of the necrotic region showed small vacuoles indicating preliminary stage of necrosis. The area of healthy cells and necrotic cells were located in the same lobular region. Necrotic region showed the pathological architecture as described above and in the region of healthy cells normal histological structure was evident. Centrolobular region of rats treated with abhrak bhasma (20 mg) along with CC14 showed normal

1024 INDIAN J EXP BlOL, OCTOBER 2001

cellular architecture without any necrotic cells or cells that show any type of stress (Fig. 4). Clear bile canaliculi were noted. Distribution of Kupffer cells and sinusoidal cells was normal. The livers of rats

were totally normal when treated with abhrak bhasma (30 and 40 mg /kg body wt) concomitant with CC14 .

The histological alterations have clearly indicated that 20 mg of abhrak bhasma protects liver against CC14

Figs 1-4----(1) Section of normal rat liver showing normal architecture. (2) Group rr rat liver showing centrolobular necrosis due to the treatment of single dose of CC1 4 . (3) Fig. 3 demonstrates reduction of necrotic area, increased number of hepatocytes, partially protected hepatocytes and bile canaliculi after CC14 + 10 mg abhrak bhasma treatment to Group Ill rats. (4) Group IV (CC1 4 + 20 mg abhrak bhasma treated rat) presents normalised picture of centrolobular region of liver.

BUWA et al.: HEPATOPROTECTIVE ACTION OF ABHRAK BHASMA 1025

induced centrolobular necrosis. Similar type of hepatoprotection against CC14 was also noted by tamra bhasma2 1

.

The histologal architectures of rat kidneys were not altered and were normal after administrations of single dose CC14 or different doses of abhrak bhasma along with CCI4.

Table 1 represents the alterations in wet weights of liver, kidney and adipose tissue of rats. Abhrak bhasma counteracted the action of CC14 on the weights of liver. Kidney weights were not altered by CC14 or CC14 + different doses abhrak bhasma. Simi larly wet weights of adipose tissues were not changed during present study.

The data on the alterations in lipolytic activities of rat liver, kidney and adipose tissues are given in Tables 2 to 4.

Carbon tetrachloride treatment causes accumula­tion of lipids in the liver and kidney8

. It is suggested that lipids from peripheral adipose tissue are

translocated to liver and kidney for accumulation8. It

has been also shown in this laboratory that lipases play important role in hepatic necrosis5

.7

'2 1

. In the present study, it was observed that, acid and alkaline lipases of liver were found to be reduced after the administration of single dose of CC14 . Similar fall in acid lipase and accumulation of triacylglycerol were noted in rat hepatocytes in primary culture in the presence of CCI/ 2

. Reduction in alkaline lipase indicated the damage to ER, since it is bound to ER4

.

Higher activity of liver lipoprotein lipase may indicate increased secretion of lipoproteins after the adminis­tration of CC14. This increase may be attributed to the stimulation of tissue repair after 24 hr of CC14

injection . Both the damage and recovery proceed simultaneousl/3

. However, it is inhibited by repeated doses of CCI/ Progressive histological recovery and enhanced acid/lysosomal lipase activity of liver by oral administration of increasing doses of abhrak bhasma concomitant with CC14 indicated the degra-

Table 1-Abhrak bhasma mediated variations in the wet weights (mg/g body wt) of liver, kidney and adipose ti ssue of albino rat during hepat iti s induced by single dose of CCI4

[Values are mean ± SE of 8 animals]

Group Li ver Kidney Adipose tissue

Gr I 33.32 ± 1.68 6.70 ± 0.39 6.79 ± 0.35

Grll 39.89 ± 1.92c 6.67±0.4l d 6.33 ± 0.47''

Gr lii 30.63 ± 1.83"· g 6.16 ± 0.51"" 0 6.04 ± 0.33b. c

GrlV 30.9 1 ±2.06c. h 6.08 ± 0.43"· c 6.06 ± 0.45b. c

GrV 30.79 ± 2.18c. h 6.18 ± 0.38"· 0 6.70 ± 0.44d. r

Gr VI 30.52 ± 1.63c. h 6.42 ± 0.31 d. g 7.36 ± 0.52d. e

P values-" <0.05; b <0.0 I ; c <0.00 I ; d >0.05; e <0.05; r <0 .01, g <0.00 I & 11 >0.05 Gr I - Normal; Gr II - CCI4 ; Gr III -CCI4 + Abhrak bhasma ( 10 mglkg body wt); Gr IV -CC I4 + Abhrak bhasma (20 mg/kg body wt) ; Gr V-CCI4 + Abhrak bhasma (30 mg/kg body wt); and Gr VI-CCI 4 + Abhrak bhasma (40 mg/kg body wt)

Table 2-Effect o f abhrak bhasma on lipolyti c enzymes of liver during hepatiti s induced by single dose of CCI 4

[Values are mean± SE of 8 animal s]

Group Acid lipase Alkaline lipase

A B A B

Gr I 10. 15 ± 3.33 47.34 ± 2.80 I 9.50 ± 1.27 90.97 ± 3.7 1

Gr ii 4.10±0.17c 16. 10 ± 0.74c 8.05 ± 0.42c 31.62 ± 1.44c

Gr lli 5.20 ± 0. 36c. g 28.74 ± 1.85c. g 10.45 ± 0.4lb.g 55.20 ± 2.86c. g

Gr IV 8.65 ± 0.53c. g 53.79 ± 2.74b. g 15 .55 ± 0.73c.g 96.60 ± 3.47a. g

GrV 12.50 ± 0.57 11• g 60. 16 ± 2.56c. g 17.75 ±0.94b.h 85.43 ± 3. 12"· g

GrVI 9.00 ± 0.44"· g 57.73±2.19b. g 19.10 ±0.85d.f 122.51 ± 4.07c.g

P values are as in Table I. A - K Units/g tissue; B - Units/mg protein Gr I, Gr II , GrIll , Gr IV, Gr V and Gr VI as in Table 1.

Lipoprotein lipase

A B

2.50 ± 0.08 11 .66 ± 0.58

6.70 ± 0.39c 26.32 ± 1.92c

8.50 ± 0.34c.f 46.98 ± 2.88c.g

11.25 ± 0.5 I e.g 69.96 ± 4.27c.g

27 .50 ± I .83c.g 132.35 ± 6.35c.g

3 1.00 ± 2.03c.g 198.85 ± 8.ooc.g

1026 INDIAN J EXP BIOL, OCTOBER 2001

Table 3--Effect of abhrak bhasma on lipolytic enzymes of rat kidney during hepatitis induced by single dose of CCI4

[Values are mean± SE of 8 animals]

Group Acid lipase Alkaline lipase Lipoprote in lipase

A B A B A B

Gr I 8.60 ± 0.43 67.56 ± 4.09 10.50 ± 0.40 82.48 ± 7.90 16.50 ± 0.071 129.62 ± 6.79

Grll 3.60 ± 0.16c 29.85 ± 1.16c 5.00 ± 0.25c 41.46 ± 2.00c. g 4.70 ± 0.17c 38.97 ± 3.05 c

Gr III 6.60 ± 0.33 b. g 49.29 ± 3.33c. g 3.80 ± 0.09c.g 28 .38 ± 1.04c. g 24.10± 1.09c.g 179.99 ± 8.38c.g

GriV 7.75 ± 0.47"·g 52.58 ± 4.18c. g 6.65 ± 0.4J c.g 45.12 ± 3.26c. c 25.14 ± 1.63c. g 170.67 ± 7.73c.g

GrV 16.00 ± 0.92c. g 66.33 ± 3.92d. g 8.00 ± 0.39c. g 33.17 ± 1.64c.g 23.56 ± 0.96c. g 97.66 ± 5.07c.g

Gr VI 18.50 ± 1.07c. g 120.05 ± 5.48c. g I 0.05 ± 0.49a g 65.22 ± 3.27C, g 24.45 ± 1.45c.g 158.45 ± 6.14c. g

P values are as in Table I. A- K Units/g ti ssue; B - Units/mg protein Gr I, Gr II, Gr III , Gr IV, Gr V and Gr VI as in Table I.

Table 4--Abhrak bhasma mediated changes in lipases of rat adipose ti ssue during hepatiti s induced by single dose of CCI4

[Values are mean± SE of 8 animals)

Group Acid lipase Alkaline lipase Lipoprotein lipase Hormone sensitive lipase

A B A B A B A B

Grl 2.1 0 ± 0.06 39.18± 1.74 4.65 ± 0.20 86.75 ± 4.31 2.50 ± 0.12 46.64 ± 2.37 8. 10 ± 0.44 151.12 ± 6.38

Grll 1.60± 1.04' 31.84 ± 1.46' 12.50 ± 0.55' 248.76 ± 14.84' 26.90 ± 0.87' 535.32 ± 28.00' 38.25 ± 12.05' 761.79 ± 34.00'

Grill 4.25 ± 0.21 '·• 105.72 ± 4.81 '·' 18.00 ± 0.86'·' 447.76 ± 21.51 '·' 24.40 ± 1.06'·' 606.97 ± 40.28'·' 23.75 ± 2.03' ·' 590.7 1 ± 31.31 '·•

GriY 4.00 ± 0. 13'·• 49.74 ± 1.92'·' 15.05 ± 0.67' · ' 187. 14 ±9.15'·' 20.70 ± 0.84'·• 257.40 ± 12.41 '·• 14.05±0.94'· ' 174.7 1 ± 9.03'·•

Gr Y 3.05 ± 0.17'·• 46.02 ± 2.33'· g 8.75 ± 0.48'·• 132.02 ± 6.51 '·. 16.25 ± 0. 77'·' 245 .17 ± 14.00'·• 11 .90 ± 0.48'· . 179.54 ± 7.77'·•

GrYI 2.35 ± 0.15'·. 43.84 ± 3. 10'·• 6.88 ± 0.36'·. 128.00 ± 5. 13'·• 12.75 ± 0.53'·' 237.87 ± I 0.89'··' 6.00 ± 0.43'·• 111.94 ± 5.86'·•

P values are as in Table I. A - K Units/g ti ssue; B - Units/mg protein Gr I, Gr II , GrIll , Gr IV. Gr V and Gr VI as in Table I

dation of accumulated lipids during hepatoprotection (which are translocated due to CC14 toxicity) . Linear rise in acid lipase activity was noted in groups 3 to 5 and declined in group 6 rat liver. These results suggest that during progressive hepatoprotection acid lipase activity increased and during total hepato­protection it tended towards normal value. Rise in alkaline lipase activity as a function of dose of abhrak bhasma suggested the recovery of endoplasmic reticulum that was supported by histological recovery at 20 to 40 mg of abhrak bhasma. Lipoprotein lipase activities exhibited the alterations parallel to alkaline lipase activities suggesting the improvement in lipo­protein metabolism.

All lipases of group 2 rats' kidneys were suppres­sed by CC14 treatment deteriorating lipid metabolism. Similar observations were noted earlier after the administration of CC14 in liquid paraffin for 11 days5

.

Administration of increasing doses of abhrak bhasma raised all lipases. Higher acid lipase actiVIty suggested increased degradation of cellular accumu-

lated lipids. However, rise in alkaline lipase activities and lipoprotein lipase activities after abhrak bhasma treatment along with CC14 indicated higher uptake and turnover of lipids to overcome the stress induced by CC14• This can be attributed to high-energy turnover in kidney during hepatoprotection.

Acid lipase activity of adipose tissue was inhibited by administration of CC14 . While the activities of alkaline, lipoprotein and hormone sensitive lipases were higher at 24 hr after the injection of CC14

suggesting the mobilisation of lipids from adipose tissue for accumulation during CC14 toxicity. Similar observations were noted in our earl ier work in this laboratori -7 . The present results indicated high turnover of lipids after 24 hr of CCI 4 injection. All lipases increased in groups 3 and 4 rat adipose tissues and brought towards normal values (although they were higher than normal values) in groups 5 and 6 in response to higher doses of abhrak bhasma. Alterations in wet weight and lipase activities suggested that during hepatoprotection turnover of

BUWA et al.: HEPATOPROTECTIYE ACTION OF ABHRAK BHASMA 1027

lipids occurs, while as the liver recovery progresses the lipid turnover declines. All lipases exhibit similar behaviour after the administration of increasing doses of abhrak bhasma concurrent with CCl4.

Abhrak bhasma is an amorphous powdery ayurvedic drug prepared from mica (which may be an organometallic complex of mica) . Mica mainly contains silicates of iron, magnesium and aluntinum 13

.

In recent years silicon and silicates have been used for the treatment of diseases. Howard and Lloyed24 have shown the behaviour of silicate nanocolloid as free radical scavenger in vivo. Abhrak bhasma might be behaving as free radical scavenger and reducing ER damage and protecting liver and kidney against CCl4 toxicity .

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