Results And Discussion A Chemistry Biology Essay

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Compound I (Lupeol) : m.p. 213 -215°C (lit 215°C)26 gave characteristic colour reaction for triterpenes. It gave a red colour in Salkowski's test and a green colour with Libermann Burchardt's Test. The presence of characteristic diagnostic absorption of vinylidine (>C=CH2) at 879.5 cm-1 indicated the possibility of lupane skeleton. It is further supported by the presence of the wagging vibration of vinylidine hydrogen appearing strongly around 984 - 942 cm-1 and in plane bending of =CH2 at 1458.1cm-1.Further the acetate melted at 217 -219°C (lit 218°C)26. Its identity as lupeol was confirmed by direct comparison of spectral characters, by co-chromatography with an authentic sample (Sigma Chemical Company, USA).

Lupeol

Compound II (Taraxerone): m.p.236 °C gave characteristic colour reaction for triterpenes. It gave a red colour in Salkowski's test and a green colour in Liebermann Burchardt's test. The peak at 1742.37 cm-1 in IR spectra indicate C=O stretching. The proton NMR signals at δ 5.3 indicates vinylic proton. The EIMS spectrum showed the molecular ion peak at m/e 424 and M+ -H2O at m/e 406. Comparison of spectral data with the known triterpenoid supported its characterization as taraxerone72.

Taraxerone

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Compound III (Acacetin) : m.p.320 - 322°C gave positive Shinoda's test. The nature of acacetin as flavone was determined on the basis of UV absorption in the region of 269 nm. ( band-I) and 327 nm ( band - II). The presence of free 7 - OH group was indicated by NaOAc induced shift of 8 nm in ring A. The UV spectral values for isolated flavone led to the conclusion that the compound is acacetin. The values for IR spectrum also in good agreement in those of acacetin reported73.

Acacetin

Compound IV ( Chrysoeriol): m.p. 325 - 327°C gave positive Shinoda's test. The nature of Chrysoeriol as flavone was determined on the basis of UV absorption in the region of 269 nm (band- I) and 327 nm (band - II). The presence of free 7-OH group was inferred by NaOAc induced bathochromic shift of 6 nm to 20 nm ( of band - II). The presence of OH groups in ring A and ring B was inferred by sodium methoxide induced bathochromic shift at both band - I and band - II. The presence of 5 -OH, 4 - Keto group was determined by using aluminium chloride which showed band - I bathochromic shift of 35 to 55 nm. The proton NMR signals at δ 3. 894 accounting for 3 protons indicated the presence of - OCH3 group. Signals at δ 9.942, δ 10.804 and δ 11.964 indicated the protons of C4'- OH, C7 - OH and C5 - OH group. Since chrysoeriol is an 3' , 4' - dioxygenated flavonoid, the C - 5' proton appeared as a doublet in the region. δ 6.7 to δ 7.1 p.p.m. The signals of the C - 2ï‚¢ and C - 6ï‚¢ protons ( which often overlap) occurred between δ 7.2 and δ 7.9 p.p.m. The EIMS spectrum showed the M+ +2 at 302, the M+ - CH3 at 285 and M+ - CO at 268. The acetate derivative prepared gave the melting point 218 - 220°C. Comparison of the spectral data with the known flavones supported its characterization as chrysoeriol74.

Chrysoeriol

Compound V (chrysoeriol - 6'' (OAc)- 4' - β- glucoside): m.p. 248 °C answered the Shinoda test for flavones and Molisch test for glycoside indicating a flavone glycoside. The paper chromatography of the hydrolysis remnant of the compound revealed that it is a glycoside of compound IV (chrosoeriol) and glucose. When UV spectrum (NaOMe) was compared with that of compound IV (chrysoeriol), no shift was observed in the band - I indicating the glycosylation site at C4'. Free hydroxyl group at C7 is indicated by the bathochromic shift of band - II in UV (NaOAc) in comparison with compound IV (chrysoeriol). The peak at 1651.9 cm-1 in IR spectra indicates the chelated C= O structure, 3081.1

cm-1 indicates the α, β - unsaturated C = C sturucture. The proton NMR signals at δ 2.004 accounted for 3 protons (CH3CO) of acetyl group. The absence of signal at δ 9.942 for C4' -OH in compound IV (chrysoeriol) clearly indicated the glycosylation at C4' . This was further confirmed in the 13CNMR, where the signal at δ 181.261 indicating C=O carbon. The EIMS spectra indicated the peaks at 507 (M+ +2), 487 (M+ - H2O) and 426 (M+ - H2O - OAc). These spectral data confirmed the acetyl glycoside nature of compound. The 6'' OH group of glucose is found to be acetylated in comparison with the 13CNMR signal at δ 62.736 given for C6'' acetylated carbon in the literature75. Comparison of the spectral data with the known flavones supported its characterization as chrysoeriol - 6'' (OAc)- 4' - β- glucoside 76.

chrysoeriol - 6'' (OAc)- 4' - β- glucoside

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Compound VI (α-amyrin acetate): m.p. 227°C (lit. 227°C)27 gave characteristic colour reaction for triterpenes. It gave a green colour with Liebermann Burchardt's test. An ester linkage was discernible from the characteristic IR adsorption at 1735.8cm-1. The acetate nature of the ester was indicated by the characteristic acetyl protons singlet at δ 2.069. Further more mass fragment m/z 408 is suggestive of the loss of acetic acid (60) from the molecular ion (M+) 468. The peaks at 250 and 218 (Base peak) m/z were due to Retro-Dials -Alder fragmentation77 with the usual hydrogen transfer, characteristic of the left and right half arising from triterpene having δ12-oleanane /ursane structure. The peak at 190 (250-60) m/z was due to loss of acetate moiety from the left half providing conclusive proof for the attachment of the acetate grouping at C-3.

The fact that it is the acetate of α amyrin was established by saponification leading to the isolation of the triterpene alcohol, melting point of which (184°C) was in good agreement with that reported for α-amyrin (lit 186 °C)27.

 amyrin acetate

Compound VII (β-Sitosterol): m.p. 138 - 140 °C (lit 140°C)28. It gave a characteristic colour reaction for a sterol. The yellow colour obtained with tetranitromethane confirmed unsaturation in the molecule. Its acetate melted at 126-128°C (lit 127-128°C)28. 13CNMR spectral data matched exactly with that of β-Sitosterol75. The most downfield signals at δ145 was accommodated for sp2 (olefinic) carbon at C-5 and the next downfield signal at δ121 to C-6 carbon. The oxigentated carbon at C-3 gave a downfield signal at δ 78 ppm. The next downfield at δ56 was accommodated for C-17. Other carbon atoms of the steroidal skeleton except that in the side chain appeared in the range

δ45-δ30 ppm. The angular methyl groups and the side chain methyl carbons gave signal in the region δ19.8 - δ 18.24 ppm. Its identity as β-sitosterol was further confirmed by IR, 1HNMR spectral characters and by co-chromatography with an authentic sample (Sigma Chemical Co., USA).

β-Sitosterol

Compound VIII (Stigmasterol): m.p. 167 -170°C (lit 170°C)29 showed positive response to Liebermann Burchardt's test and Salkowski's test. It formed an acetate m.p. 143 -145°C (lit 144°C)32. 13CNMR spectral data matched exactly with that of Stigmasterol78. The most downfield signals at δ145 was accommodated for sp2 (olefinic) carbon at C-5 and the next downfield signal at δ138 ppm and 128 ppm to C-22 and C-23. The downfield signal at δ 121 is to C-6. The oxygenated carbon at C-3 gave a downfield signal at δ78 ppm. The next downfield signal at δ56 ppm was accommodated for C-17. Other carbon atoms of the steroidal skeleton except that in the side chain appeared in the range δ 45- δ 30 ppm. The angular methyl groups and the side chain methyl carbons gave signals in the region δ 19.8 - δ 18.24 ppm. Its identity as stigmasterol was further confirmed by IR, 1HNMR spectral characters and by co-chromatography with an authentic sample (Sigma Chemical Co., USA).

Stigmasterol

Compound IX (β-Sitosterol): m.p. 138 - 140 °C (lit 140°C)28. It gave a characteristic colour reaction for a sterol. The yellow colour obtained with tetranitromethane confirmed unsaturation in the molecule. Its acetate melted at 126-128°C (lit 127-128°C)28. Its identity as β-sitosterol was confirmed by spectral characters and by co-chromatography with an authentic sample (Sigma Chemical Co., USA).

Compound X ( Phyllanthin):m.p. 96°C (lit 98°C)79 gave bluish green colour with MeOH/H2SO4 ( 9:1). 13CNMR spectral data matched exactly with that of phyllanthin80. The most downfield signal at δ 148.7 ppm and δ 147.9 ppm was accommodated for oxygenated aromatic carbon at C -3, 3', 4 and 4'. The next down field signal at δ 133.57 ppm was accommodated for C- 1 and 1' . the aromatic carbon atoms of the compound appeared in the range δ133.57 to δ 111.05. The aliphatic carbon atoms at C-8 and 8΄ and also at C - 7 and 7' appeared at δ 40.65 and δ 34.83. Comparison of the spectral data with the known lignan supported its characterization as phyllanthin80.

Phyllanthin

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Compound XI (Hypophyllanthin): m.p. 128 °C ( lit 128°C) gave violet colour with MeOH/H2SO4 (9:1). The IR spectrum contains a strong band at 939.3 cm-1 which is a characteristic of methylenedioxy group. The 1HNMR spectrum showed peak at δ 5.65 suggesting the presence of methylenedioxy group. 13CNMR spectral data matched exactly with that of hypophyllanthin80. The most downfield signal in the range δ 142.82 to δ 149.35 was accommodated for oxygenated aromatic carbon at C -3' ,4', 8,7 and 6. Other aromatic carbon atoms of compound appeared in the range δ 107.38 to δ 111.57. Comparison of the spectral data with the known lignan supported its characterization as hypophyllanthin80.

Hypophyllanthin

B. Pharmacology

Hepatoprotective activity

Effect of the petroleum ether, ether and ethyl acetate fractions of the ethanol extract of the aerial parts of Leucas lavandulaefolia on CCl4 induced hepatotoxicity in male albino rats( table 7)

Acute toxicity study

The petroleum ether, ether and ethyl acetate fractions of the ethanolic extract did not exhibit any toxic effects upto 4000 mg /kg body weight when administered as single oral dose.

Selection of dose

The LD50 of the petroleum ether, ether and ethyl acetate fractions of the ethanol extract was more than 4000 mg/kg body weight in all the cases. Therefore 1/10th of maximum tolerated dose i.e. 400 mg/kg body weight was chosen for the study.

Group I

Biochemical studies

The serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) levels stood at 45.41± 1.11 and 45.13± 1.23 U/L respectively. Whilst the alkaline phosphatase level was 115.31± 1.35 U/L (table 7).

Histopathological studies

The histopathological picture showed normal structure of hepatic cells. Section showed structure of liver whose architecture is well preserved. The hepatocytes are normal. The sinusoids shows a fenestrated endothelial cell lining. The central veins appear normal and the hepatocytes, appear to be arranged radiating away from it. The portal tract composed of portal vein radical. However no liver cell injury/necrosis is seen (plate 5).

Group II

Biochemical studies

The animals of this group displayed a significant increase in SGOT (155.5± 1.44 U/L) and SGPT (140.12 ± 2.22 U/L) and alkaline phosphatase (190.33 ± 1.04 U/L) respectively, in comparison to those of the normal animals of group (table 7).

Histopathological studies

Liver exposed to CCl4 revealed the following facts. The section of liver showed marked fatty changes around portal tract and hepatocytes with fatty changes showing peripherally pushed nuclei (plate 6).

Group III

Biochemical studies

When compared to group II, this group exhibited a significant fall in SGOT (61.25± 3.61 U/L) and SGPT (62.33 ± 1.11 U/L) and alkaline phosphatase (143 ± 1.33 U/L) (table 7).

Histopathological studies

Liver exposed to CCl4 and treated with petroleum ether extract of aerial parts of L. lavandulaefolia showed almost complete restoration of normalcy. There is neither degeneration nor necrosis of hepatocytes noted. More over, most of the portal tracts appeared normal. Occasional binucleate cells seen focally suggesting regeneration( plate 7).

Group IV

Biochemical studies

The SGOT (51.3 ± 1.32 U/L), SGPT ( 57.11± 1.22 U/L) and alkaline phosphatase ( 120.1 ± 1.30 U/L) decreased significantly when compared to CCl4 treated animals of group II( table 7).

Histopathological studies

Liver exposed to CCl4 and treated with ether extract of aerial part of L. lavandulaefolia revealed normalcy of hepatic cells, central vein and portal vein (plate 8).

Group V

Biochemical studies

The SGOT (46.5 ± 0.55 U/L) and SGPT ( 46.61 ± 2.68 U/L) and alkaline phosphatase (118.22 ± 2.63 U/L) decreased significantly when compared to the CCl4 treated animals of group II ( table 7).

Histopathological studies

Liver exposed to CCl4 and treated with ethyl acetate extract of aerial part of L. lavandulaefolia revealed normalcy of hepatic cells, central vein and portal vein (plate 9).

Table 7 Effect of various fractions of the ethanol extract of the aerial parts of L. lavandulaefolia on CCl4 induced biochemical and histopathological changes in male albino rats

Groups

(n =6)

SGOT

(U/L)

SGPT

(U/L)

ALP

(U/L)

Histopathology

D

N

Group I

(Normal)

44.41± 1.11

45.13 ± 1.23

115.31± 1.35

0

0

Group II

(CCl4)

155.5± 1.44*

140.12±2.22*

190.33 ±1.04*

+++

+++

Group III

( petroleum ether)

61.25 ± 3.61*

62.33 ± 1.11*

143 ± 1.33 *

0

0

Group IV

(ether)

51.3 ± 1.32*

57.11 ± 1.22 *

120.1 ± 1.30 *

0

0

Group V

(ethyl acetate)

46.5 ± 0.55*

46.61 ± 2.68 *

118.22 ± 2.63*

0

0

* P < 0.001

Values are mean ± SEM, of six animals in each group. Data was analysed by unpaired 't' test.

n = number of animals in each group D = degeneration N = necrosis

Effect of the petroleum ether fraction of the ethanol extract of the aerial parts of Phyllanthus lawii on CCl4 induced hepatotoxicity in male albino rats (table 8).

Acute toxicity study

The petroleum ether fractions of the ethanolic extract did not exhibit any toxic effects upto 4000 mg /kg body weight when administered as single oral dose.

Selection of dose

The LD50 of the petroleum ether fraction of the ethanol extract was more than 4000 mg/kg body weight in all the cases. Therefore 1/10th of maximum tolerated dose i.e. 400 mg/kg body weight was chosen for the study.

Group I

Biochemical studies

The serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) levels stood at 47.11± 2.65 and 49.02± 22 U/L respectively. Whilst the alkaline phosphatase level was 118± 2.11 U/L (table 8).

Histopathological studies

The histopathological picture showed normal structure of hepatic cells section showed structure of liver whose architecture is well preserved. The hepatocytes are normal (plate 10).

Group II

Biochemical studies

The animals of this group displayed a significant increase in SGOT (156.5± 1.52 U/L) and SGPT (145.12 ± 1.31 U/L) and alkaline phosphatase (192.31 ± 1.69 U/L) respectively, in comparison to those of the normal animals of group (table 8).

Histopathological studies

Liver exposed to CCl4 revealed the following facts. The section of liver showed marked fatty changes around portal tract and hepatocytes with fatty changes showing peripherally pushed nuclei (plate 11).

Group III

Biochemical studies

When compared to group II, this group exhibited a significant fall in SGOT (49.11± 3.11 U/L) and SGPT (51.12 ± 2.23 U/L) and alkaline phosphatase (122.36 ± 2.11 U/L) (table 8).

Histopathological studies

Liver exposed to CCl4 and treated with petroleum ether extract of the aerial part of P. lawii revealed normalcy of hepatic cells, no fatty changes and necrosis (plate 12)

Table 8 : Effect of petroleum ether fraction of the ethanol extract of the aerial parts of P. lawii on CCl4 induced biochemical and histopathological changes in male albino rats

Groups

(n =6)

SGOT

(U/L)

SGPT

(U/L)

ALP

(U/L)

Histopathology

D

N

Group I

(Normal)

47.11± 2.65

49.02 ± 2.22

118.33± 2.11

0

0

Group II

(CCl4)

156.5± 1.52*

145.12 ± 1.31*

192.31 ± 1.69*

+++

+++

Group III

( petroleum ether)

49.11 ± 3.11 *

51.12 ± 2.23 *

122.36± 2.11*

0

0

* P < 0.001

Values are mean ± SEM, of six animals in each group. Data was analysed by unpaired 't' test

n = number of animals in each group D = degeneration N = necrosis.

Effect of petroleum ether extract of whole plant of Phyllanthus debilis on CCl4 induced hepatotoxicity in male albino rats ( table 9)

Acute toxicity study

The petroleum ether extract did not exhibit any toxic effects upto 4000 mg /kg body weight when administered as single oral dose.

Selection of dose

The LD50 of the petroleum ether extract was more than 4000 mg/kg body weight in all the cases. There for 1/10th of maximum tolerated dose i.e. 400 mg/kg body weight was chosen for the study.

Group I

Biochemical studies

The serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) levels stood at 46.31± 4.11 and 48.50± 2.01 U/L respectively. Whilst the alkaline phosphatase level was 119.13± 2.14 U/L (table 9).

Histopathological studies

The histopathological picture showed normal structure of hepatic cells. Section showed structure of liver whose architecture is well preserved. The hepatocytes are normal (plate 13).

Group II

Biochemical studies

The animals of this group displayed a significant increase in SGOT (151.21± 2.39 U/L) and SGPT (148.11 ± 2.33 U/L) and alkaline phosphatase (198.42 ± 3.11 U/L) respectively, in comparison to those of the normal animals of group (table 9).

Histopathological studies

Liver exposed to CCl4 revealed the following facts. The section of liver showed marked fatty changes around portal tract and hepatocytes with fatty changes showing peripherally pushed nuclei (plate 14).

Group III

Biochemical studies

When compared to group II, this group exhibited a significant fall in SGOT (53.11± 4.21 U/L) and SGPT (52.49 ± 3.65 U/L) and alkaline phosphatase (120.11 ±3.52 U/L) (table 9).

Histopathological studies

Liver exposed to CCl4 and treated with petroleum ether extract of the whole plant of P. debilis revealed normalcy of hepatic cells. The hepatocytes showed 2 to 3 nuclear suggestive of regenerative activity

(plate 15).

Table 9 Effect of petroleum ether extract of whole plant of P. debilis on CCl4 induced biochemical and histopathological changes in male albino rats

Groups

(n =6)

SGOT

(U/L)

SGPT

(U/L)

ALP

(U/L)

Histopathology

D

N

Group I

(Normal)

46.31± 4.11

48.50 ± 2.01

119.13± 2.14

0

0

Group II

(CCl4)

151.21±2.39*

148.11 ±2.33*

198.42 ±3.11*

+++

+++

Group III

( petroleum ether)

53.11 ± 4.21 *

52.49 ± 3.65 *

120.11± 3.25 *

0

0

* P < 0.001

Values are mean ± SEM, of six animals in each group. Data was analysed by unpaired 't' test

n = number of animals in each group D = degeneration N = necrosis

Anti inflammatory activity

Effect of petroleum ether, ether and ethyl acetate fractions of the ethanol extract of the aerial parts of L. lavandulaefolia on carrageenan induced inflammation in albino rats

Acute toxicity study

The petroleum ether, ether and ethyl acetate fractions of the ethanolic extract did not exhibit any toxic effects upto 4000 mg /kg body weight when administered as single oral dose.

Selection of dose

The LD50 of the petroleum ether, ether and ethyl acetate fractions of the ethanol extract was more than 4000 mg/kg body weight in all the cases. Hence a dose of 100, 200 and 400 mg/kg body weight were chosen for the study.

In carrageenan induced rat hind paw method, statistically it was found that petroleum ether, ether and ethyl acetate extract of aerial parts of L. lavandulaefolia , at a dose of 400 mg/kg body weight significantly reduced the oedema volume, which was comparable to standard drug diclofinac sodium. The petroleum ether, ether and ethyl acetate extract of the aerial part of L .lavandulaefolia also showed dose dependent anti inflammatory activity. The results are depicted in table 10.

Table 10 Anti inflammatory activity of the petroleum ether, ether and ethyl acetate extracts of aerial parts of L. lavandulaefolia

Treatment

Dose

(mg/kg p.o.)

Oedema volume

(ml)

Inhibition

(%)

Control (Tween 80, 1%)

……

0.16 ± 0.010

……

Petroleum ether extract

100

0.13 ± 0.007 **

18.75

200

0.09 ± 0.003*

43.75

400

0.06 ± 0.012 *

62.50

Diethyl ether extract

100

0.13 ± 0.004**

18.75

200

0.09 ± 0.014*

43.75

400

0.06 ± 0.005 *

62.50

Ethyl acetate extract

100

0.14 ± 0.010**

12.50

200

0.08 ± 0.006*

50.0

400

0.06 ± 0.011 *

62.50

Diclofinac sodium

100

0.05 ± 0.010*

68.80

*p < 0.001 compared to control

** p < 0.01 compared to control

Values are mean ± SEM of six animals in each group. Data was analysed by unpaired 't' test

Effect of petroleum ether fraction of the ethanolic extract of the aerial parts of Phyllanthus lawii on carrageenan induced inflammation in albino rats

Acute toxicity study

The petroleum ether fraction of the ethanolic extract did not exhibit any toxic effects upto 4000 mg /kg body weight when administered as single oral dose.

Selection of dose

The LD50 of the petroleum ether fraction of the ethanol extract was more than 4000 mg/kg body weight in all the cases. Hence a dose of 100, 200 and 400 mg/kg body weight were chosen for the study.

In carrageenan induced rat hind paw method, statistically it was found that petroleum ether extract of aerial parts of P. lawii, at a dose of 400 mg/kg body weight significantly reduced the oedema volume, which was comparable to standard drug diclofinac sodium. The petroleum ether extract of the aerial part of P. lawii also showed dose dependent anti inflammatory activity. The results are depicted in table 11.

Table 11 Anti inflammatory activity of the petroleum ether extract of aerial parts of P. lawii

Treatment

Dose

(mg/kg p.o.)

Oedema volume

(ml)

Inhibition

(%)

Control (Tween 80, 1%)

……

0.17± 0.011

……

Petroleum ether extract

100

0.14± 0.004**

17.65

200

0.10± 0.002*

41.18

400

0.06 ± 0.03*

64.71

Diclofinac sodium

100

0.05 ± 0.002*

70.59

*p < 0.001 compared to control

** p < 0.01 compared to control

Values are mean ± SEM of six animals in each group. Data was analysed by unpaired 't' test

Effect of petroleum ether extract of the whole plant of Phyllanthus debilis on carrageenan induced inflammation in albino rats

Acute toxicity study

The petroleum ether extract did not exhibit any toxic effects upto 4000 mg /kg body weight when administered as single oral dose.

Selection of dose

The LD50 of the petroleum ether extract was more than 4000 mg/kg body weight in all the cases. Hence a dose of 100, 200 and 400 mg/kg body weight were chosen for the study.

In carrageenan induced rat hind paw method, statistically it was found that petroleum ether extract of whole plant of P. debilis at a dose of 400 mg/kg body weight significantly reduced the oedema volume, which was comparable to standard drug diclofinac sodium. The petroleum ether extract of the whole plant of P. debilis also showed dose dependent anti inflammatory activity. The results are depicted in table 12.

Table 12 Anti inflammatory activity of the petroleum ether extract of whole plant of P. debilis

Treatment

Dose

(mg/kg p.o.)

Oedema volume

(ml)

Inhibition

(%)

Control (Tween 80, 1%)

……

0.17± 0.021

……

Petroleum ether extract

100

0.15± 0.003**

11.77

200

0.09± 0.011*

46.06

400

0.06 ± 0.041*

64.71

Diclofinac sodium

100

0.05 ± 0.011*

70.59

*p < 0.001 compared to control

** p < 0.01 compared to control

Values are mean ± SEM of six animals in each group. Data was analysed by unpaired 't' test

Analgesic activity

Effect of petroleum ether, ether and ethyl acetate fractions of ethanol extract of the aerial parts of Leucas lavandulaefolia on acetic acid induced writhing method in mice for analgesic activity.

Acute toxicity study

The petroleum ether, ether and ethyl acetate fractions of the ethanolic extract did not exhibit any toxic effects upto 4000 mg /kg body weight when administered as single oral dose.

Selection of dose

The LD50 of the petroleum ether, ether and ethyl acetate fractions of the ethanol extract was more than 4000 mg/kg body weight in all the cases. Hence 1/10th of maximum tolerated i.e. 400 mg/kg body weight was chosen for the study.

The results of acetic acid writhing test in mice showed a significant decrease in number of wriths in petroleum ether, ether and ethyl acetate extract of aerial parts of L. lavandulaefolia, suggesting peripheral analgesic effect ( table 13).

Table 13 Analgesic activity of the petroleum ether, ether and ethyl acetate extract of aerial parts of L .lavandulaefolia

Treatment

Dose

Mean writhing per

30 min± SEM

Inhibition

(%)

Control

Tween 80, p.o

88± 0.57

……

Aspirin

50 mg/kg p.o

22± 1.13*

75.00

Petroleum ether extract

400 mg/ kg p.o

33± 1.15*

62.50

Ether extract

400 mg/ kg p.o

32 ± 0.44*

63.64

Ethyl acetate extract

400 mg/ kg p.o

28 ± 0.25*

68.19

*p < 0.001 compared to control

Values are mean ± SEM of six animals in each group. Data was analysed by unpaired 't' test

Effect of petroleum ether fraction of ethanol extract of aerial parts of Phyllanthus lawii on acetic acid induced writhing method in mice for analgesic activity.

Acute toxicity study

The petroleum ether fraction of the ethanolic extract did not exhibit any toxic effects upto 4000 mg /kg body weight when administered as single oral dose.

Selection of dose

The LD50 of the petroleum ether fraction of the ethanol extract was more than 4000 mg/kg body weight in all the cases. Hence 1/10th of maximum tolerated i.e. 400 mg/kg body weight was chosen for the study.

The results of acetic acid writhing test in mice showed a significant decrease in number of wriths in petroleum ether extract of aerial parts of Phyllanthus lawii, suggesting peripheral analgesic effect ( table 14).

Table 14 Analgesic activity of the petroleum ether extract of aerial parts of P.lawii

Treatment

Dose

Mean writhing per

30 min± SEM

Inhibition

(%)

Control

Tween 80, p.o

85± 0.33

……

Aspirin

50 mg/kg p.o

24± 2.13*

71.77

Petroleum ether extract

400 mg/ kg p.o

29± 0.41*

65.89

*p < 0.001 compared to control

Values are mean ± SEM of six animals in each group. Data was analysed by unpaired 't' test

Effect of petroleum ether extract of whole plant of Phyllanthus debilis on acetic acid induced writhing method in mice for analgesic activity.

Acute toxicity study

The petroleum ether extract did not exhibit any toxic effects upto 4000 mg /kg body weight when administered as single oral dose.

Selection of dose

The LD50 of the petroleum ether extract was more than 4000 mg/kg body weight in all the cases. Hence 1/10th of maximum tolerated i.e. 400 mg/kg body weight was chosen for the study.

The results of acetic acid writhing test in mice showed a significant decrease in number of wriths in petroleum ether extract of whole plant of P. debilis, suggesting peripheral analgesic effect (table 15).

Table 15 Analgesic activity of the petroleum ether extract of whole plant of P. debilis

Treatment

Dose

Mean writhing per

30 min± SEM

Inhibition

(%)

Control

Tween 80, p.o

86± 0.43

……

Aspirin

50 mg/kg p.o

24± 3.22*

72.10

Petroleum ether extract

400 mg/ kg p.o

28± 1.26*

65.45

*p < 0.001 compared to control

Values are mean ± SEM of six animals in each group. Data was analysed by unpaired 't' test