Arch. Pharm. Res. DOI 10.1007/s12272-014-0484-4

RESEARCH ARTICLE

In vivo evaluation of analgesic, anti-inflammatory, and neuropharmacological activities of the chemical constituent from Nepeta clarkei Javid Hussain • Najeeb Ur Rehman • Ahmed Al-Harrasi • Abdul Latif Khan Tania Shamim Rizvi • Faryal Vali Mohammad • Mehjabeen • Liaqat Ali



Received: 26 February 2014 / Accepted: 13 September 2014 Ó The Pharmaceutical Society of Korea 2014

Abstract The plant species of genus Nepeta are used to treat various human diseases and for ornamental purposes as well. Nepethalate B (1) was isolated as a result of phytochemical investigations of Nepeta clarkei and was subjected in the present study for investigation of analgesic, anti-inflammatory, and central nervous system (CNS) depressant activities. The percentage inhibition in phase I of the acetic acid induced writhing test of compound 1 (dose of 0.1, 0.2 and 0.4 mg/kg of body weight) was 53.3, 58.0 and 86.7 % respectively. These results were found significantly higher (P \ 0.01) as compared to the negative control. Moreover, the percentage inhibitions of three phases for 0.1, 0.2 and 0.4 mg/kg were higher than the values obtained for Aspirin (positive control). In formalin test, the percentage pain inhibition between 0 and 5 min

J. Hussain (&)  N. U. Rehman  A. Al-Harrasi  A. L. Khan  L. Ali (&) Department of Biological Sciences and Chemistry, College of Arts and Sciences, University of Nizwa, Nizwa 616, Sultanate of Oman e-mail: [email protected] L. Ali e-mail: [email protected] T. S. Rizvi National Nematological Research Center, University of Karachi, Karachi 75270, Pakistan F. V. Mohammad International Center for Chemical and Biological Sciences, H. E. J. Research Institute of Chemistry, University of Karachi, Karachi 75270, Pakistan Mehjabeen Department of Pharmacognosy, Faculty of Pharmacy, Research Institute of Pharmaceutical Sciences, University of Karachi, Karachi 75270, Pakistan

(early phase) was 68.0, 25.5, and 75.5 % for 0.1, 0.2 and 0.4 mg/kg intra-peritoneal doses of compound 1 respectively. In case of late phase (20–30 min) it was 63.0, 66.7 and 48.1 %, respectively. In comparison to aspirin, overall percentage inhibition of compound 1 was significantly higher in early and late phases. Interestingly, at all doses compound 1 showed more potent anti-inflammatory effects in terms of intensity and duration as compared to aspirin. The gross behavioral study of nepethalate B (1) was also carried out and the results revealed that it exhibited CNS depression in the mice and showed a prominent decrease in locomotor activity. Keywords Nepeta clarkei  Nepethalate  Analgesic activity  Anti-inflammatory activity  Neuropharmacological activity

Introduction Nature has played an instrumental role in providing effective therapeutic entities. In fact, terrestrially derived remedies have generally preceded the progress of medicine in the context of the evolution of humankind (Jimeno et al. 2004). Serendipity sequentially linked to a scientifically oriented approach; pioneered by Paracelsus some centuries ago have led to significant discoveries in therapeutic research. In this context, it is important to consider that the major anti-infective, anticancer, analgesics and immunosuppressive compounds are of natural origin (Jimeno et al. 2004). Nepeta species are used as diuretic, diaphoretic, antitussive, antispasmodic, antiasthmatic, febrifuge, emmenagogue, sedative agents, insecticidal, acaricidal, antiviral, anti-inflammatory and antioxidant (Cigremis et al. 2010). Some of the Iranian Nepeta species

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contributed a great deal to Iranian folk and traditional medicines and are used in the treatment of various disorders such as nervous, respiratory and gastrointestinal diseases (Hussain et al. 2010). In the course of phytochemical and biological studies of medicinal plants from Pakistan (Hussain et al. 2010, 2011), we investigated analgesic, antiinflammatory, and neuropharmacological (CNS depressant) activities of nepethalate B (1), isolated (Hussain et al. 2011) from the methanolic extract of Nepeta clarkei, and the results are explained in the present paper.

J = 6.0, 2.0 Hz, 2H, H-4, H-5), 5.86 (s, 1H, H-80 , H-800 ), 4.17–4.21 (m, 2H, H-(10 ), H-(100 )), 3.80 (s, 3H, H-(100 ), H-(1000 )), 1.93–1.96 (m, 2H, H-(130 ), H-(1300 ), 1.63–1.67 (m, 6H, H-(20 ), H-(200 ), 1.39–1.41 (m, 2H, H-(40 ), H-(400 )), 1.21–1.24 (m, H-30 –60 , H-110 , H-140 ), H-300 –600 , H-1100 , H-1400 ), 0.86 (t, J = 6.0 Hz, 6H, H-120 , H-1200 ), 0.89 (t, J = 6.0 Hz, 6H, H-150 , H-1500 ). HREI-MS: m/z 642.4130 (Calcd. 642.4132 for C38H58O8). Biological evaluation Animals

Materials and methods Phytochemical investigations General Optical rotations were measured on a JASCO DIP 360 polarimeter. IR Spectra were recorded on a Bruker, VECTOR 22 spectrophotometer. EI-MS and HREI-MS were recorded on mass spectrometers JEOL JMS HX 110. The 1 H and 13C NMR Spectra were recorded on Bruker NMR spectrometers operating at 500 MHz (125 MHz for 13C). The chemical shift values are reported in ppm (d) and the coupling constants (J) are given in Hz. Plant material The plant, N. Clarkei, was collected at Parachinar, Kurram Agency, Khyber pakhtunkhwa, Pakistan, in 2005, and was identified by Mr. Muhammad Sirajh (plant taxonomist) at the Department of Botany, Govt. Jehanzeb Post Graduate College, Saidu Shareef, Swat, Pakistan. A specimen of this plant (KN-375) was deposited at the Herbarium of the department. Extraction and isolation Dried and powdered plant material of N. clarkei (6.5 kg) was extracted with MeOH and fractionated in n-hexane, CHCl3, and EtOAc. The CHCl3 fraction (55 g) was then subjected to repeated column chromatography (silica gel, n-hexane, n-hexane–CHCl3 and CHCl3–MeOH, in order of increasing polarity) yielding nepethalate B (1) (12.5 mg) by using a mixture of n-hexane–CHCl3 (3:7). Nepethalate B (1) Yellowish oil. UV (CHCl3) kmax (log e): 273 (2.85); IR mmax (CHCl3): 2924, 1720, 1604, 1570, 1480, 1265, 1115, 1070, 1030, 745, cm-1. 1H NMR (500 MHz, CDCl3): d(H) 7.68 (dd, J = 6.0, 2.0 Hz, 2H, H-3, H-6), 7.51 (dd,

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Male Swiss albino mice weighing 20–25 g were used as test animals. The animals were housed in standard cages at room temperature under controlled light and temperature with free access to food and water. Prior to the experiments (about 24 h), the animals were maintained at the laboratory conditions with free access to water only. The animals subjected to oral administration of the test samples were fasted for 12 h. Writhing test The anti-nociceptive effect was evaluated by acetic acid induced writhing test in mice. The mouse writhing test was based on the previously described method of Koster et al. (1959), Neto et al. (2005). The abdominal contortion or writhes resulting from intraperitoneal injection of 0.6 % acetic acid consist of a contraction of body muscles and the stretching of hind limb. These abdominal contortions were counted over three periods of 20 min each after injection of acetic acid. The antinociceptive activity was considered as the reduction of these abdominal contortions (writhes) in the treated animals (with test samples) as compared to control (injected only with acetic acid). The animals were pretreated orally with the test samples and aspirin (300 mg/ kg of the body weight) was used as a standard during this test. The % inhibition of the test samples was calculated by using the following formula: ðnumber of writhing of controlÞ ðno of writhing of test groupÞ= ðnumber of writhing of controlÞ  100 Formalin test The anti-nociceptive effect was also evaluated by the formalin test in mice. 2.5 % formalin solution (0.05 ml) was injected in the sub-plantar region of right hind paw of the mice to induce pain. Aspirin (36.2 % inhibition in 1st phase and 29.6 % in 2nd phase) was administered (30 min before formalin injection) orally to serve as a positive

Evaluation of analgesic, anti-inflammatory, and neuropharmacological activities

control; whereas distilled water was used as a negative control. The test samples were administered orally (300 mg/kg) 60 min before the reaction. The animals were placed in transparent Plexiglas cage observation chambers and the time spent in licking and biting responses of the injected paw was taken as indicator of pain response (de Miranda et al. 2001; Hunskaar and Hole 1987). Responses were measured for 5 min after formalin injection (early phase) and for 20–30 min after formalin injection (late phase). The reduction in the number of licking and biting responses is the inhibition of the pain score and the % inhibition can be calculated by the following expression: ðmean of control groupÞðmean of treated groupÞ= ðmean of control groupÞ  100 Anti-inflammatory studies The formalin (2.5 %) induced inflammation was used in this study, as described by Winter et al. (1962). Animals were divided into five groups of six rats each. Thirty minutes before injection of formalin, the groups were treated Intra-peritoneal thus: group 1 received 10 mg of piroxicam per kg (?ve control), group 2 received 1 ml distilled per kg (-ve control), groups 3, 4 and 5 received 25, 50 and 100 mg of extract per kg body weight,

respectively. The increase in paw diameter was measured using vernier calliper. The difference in weight of the right hind paw and the left hind paw indicates inflammation. Measurement was done immediately before and after 1–5 h following formalin injection.

CNS depressant activity The gross behavior activity was performed according to the method described by Irwin (1964). The locomotor and muscle relaxant activity (cage cross, head dip, rearing, open field, traction test, and mobility time) was evaluated according to the method described by Ahmad et al. (2011) and Verma et al. (2010). For these tests the animals were placed in specified chambers and their movements were observed for 15 min. The muscle relaxant activity was determined by placing the mice on stationary road and noting the time travelled from one end to the other; the mobility time was also noted for a period of 6 min.

Statistical analysis The statistical analyses were performed according to the method described by Alcaraz and Jimenez (1989). The values are expressed as mean ± SEM. The significance of difference between means was determined by student’s t test at P \ 0.05 and P \ 0.01 levels.

Results Compound 1 (Fig. 1) was reported earlier by our team from N. clarkei (Hussain et al. 2011) and the structure elucidation was carried out by combined 1H and 13C NMR spectral data along with 2D NMR techniques by comparison with the reported data.

Fig. 1 Structure of nepethalate B (1)

Table 1 Acetic acid induced writhing response and the inhibition of compound 1 Sample

Phase I (0–20 min) No of writhes

Phase II (20–40 min) % Inhibition

No of writhes

Phase III (40–60 min) % Inhibition

No of writhes

Control

56 ± 2.08

0.0

64 ± 2.98

0.0

13 ± 0.92

1 (0.1 mg i.p)

6.8 ± 1.22

87.8*

1.9 ± 0.08

97*

3 ± 0.07

% Inhibition 0.0 77.2*

1 (0.2 mg i.p)

4.1 ± 0.98

92.6*

1.2 ± 0.65

98*

2.1 ± 0.34

87*

1 (0.4 mg i.p)

9.2 ± 0.08

83.4*

7.2 ± 0.05

88.7*

4.8 ± 0.32

63.6*

Aspirin

18 ± 0.12

67.6

21 ± 0.45

66.8

8.2 ± 0.09

37.9

Values are mean ± SEM, n = 5, t test, the value with * are significantly different as compared to control (positive or negative) as evaluated by student’s t test (P \ 0.05)

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Analgesic activity studies

The writhing response against compound 1 was not dosedependant, but percentage inhibition of compound 1 was significantly higher in all three phases as compared to aspirin application. Although the number of writheses were more signifincant (P \ 0.05) in aspirin treatment than compound 1 but overall the different doses resulted in higher inhibition than aspirin.

The purified compound 1 (nepethalate B) was subjected to analgesic activity studies by using the following two different procedures. Writhing test The abdominal constrictions (writhes) in mice were induced by a 0.6 % acetic acid solution, when administered orally. The resulting writhes were counted for three phases, within each 0 min period up to 60 min. Aspirin (300 mg/ kg orally) was used as a positive control. The inhibition of aspirin induced writhing was observed up to 67.6 % in phase I, 66.8 % in phase II, and 37.9 % in phase III. The percentage inhibition of compound 1 at 0.1, 0.2 and 0.4 mg/kg of body weight (intraperitoneally) in phase I was found to be 29.8, 36.9 and 23.3 % respectively higher than aspirin control. This was significant (P \ 0.01) when compared to positive control. Moreover, the percentage inhibitions of all three phases are summarized in Table 1.

Formalin test The results in Table 2 demonstrate the significant antinociceptive effect of compound 1 in both phases of formalin test. Compound 1 (orally administered) inhibited significantly the licking and biting response, induced by formalin in the biphasic pain response (the neurogenic pain response or the early phase and the inflammatory pain response or the late phase). The nepethalate B (1) extract dose-dependently reduced formalin-induced pain in both early and late phases of the experiment. However, the percentage pain inhibitions between 0 and 5 min (early phase) were 68.0, 25.5, and 75.5 % for 0.1, 0.2 and 0.4 mg/kg intra-peritoneal doses of compound 1, respectively, while the percentage pain inhibitions between 20 and 30 min (late phase) were 63.0, 66.7 and 48.1 %, for 0.1, 0.2 and 0.4 mg/ kg intra-peritoneal doses of compound 1, respectively (Table 2). The results were significantly (P \ 0.05) different from the values obtained for the negative control group. When compared with positive control (aspirin), compound 1 at 0.1 and 0.4 mg/kg showed a higher percentage inhibition (68.0 and 75.5 %) than aspirin in early phase. Similarly compound 1 also showed better percentage inhibition than aspirin at 0.1, 0.2 and 0.4 mg/kg in late phase (63.0, 66.7 and 48.1 % respectively). In early and late phases of formalin induced licking and biting responses, the values of 0.1 and 0.4 mg i.p. were significantly higher (P \ 0.05) against compound 1 as compared to aspirin.

Table 2 Percentage inhibition of compound 1 via Formalin induced licking and biting responses Samples

Early phase (0–5 min)

Late phase (20–30 min)

No of licking and biting

No of licking and biting

% Inhibition

% Inhibition

Control

47 ± 2.08

0.00

27 ± 2.98

0.00

1 (0.1 mg i.p)

15 ± 1.22

68.0*

10 ± 0.08

63*

1 (0.2 mg i.p)

12 ± 0.98

25.5

09 ± 0.65

66.7*

1 (0.4 mg i.p)

20 ± 0.13

75.5*

14 ± 0.07

48.1*

Aspirin

30 ± 0.12

36.18

19 ± 0.45

29.63

Values are mean ± SEM, n = 5, t test, P \ 0.05; the value with * are significantly different as compared to control (positive or negative) as evaluated by student’s t test (P \ 0.05)

Table 3 Anti-inflammatory activity of compound 1 in rat Treatment (mg/kg)

0

0.25/15 min

0.30/30 min

1st hour

2nd hour

3rd hour

4th hour

12th hour

Control

0.9 ± 0.13

1.3 ± 0.08

1.45 ± 0.07

1.55 ± 0.08

1.6 ± 0.02

1.7 ± 0.05

1.82 ± 0.08

1.3 ± 0.67

0.1 mg (I/P)

1.1 ± 0.15

1.25 ± 0.12

1.25 ± 0.27

1.32 ± 0.07

1.29 ± 0.02

1.35 ± 0.05

1.32 ± 0.06

0.9 ± 0.03

% Inhibition

11.1

7.7

13.8

14.8

19.4

20.6

27.5

30.8

0.2 mg (I/P)

0.85 ± 0.25

1.22 ± 0.02

1.23 ± 0.17

1.28 ± 0.25

1.27 ± 0.13

1.33 ± 0.12

1.32 ± 0.13

0.9 ± 0.13

% inhibition

5.6*

7.7

15.2*

17.4*

20.6*

21.8*

27.5*

30.8

Aspirin 300 mg/kg

0.92 ± 0.25

1.1 ± 0.18

1.32 ± 0.17

1.35 ± 0.05

1.37 ± 0.12

1.45 ± 0.15

1.42 ± 0.09

0.9 ± 0.75

% Inhibition

2.22

15.39

8.97

12.91

14.38

14.71

21.98

30.77

Values are mean ± SEM, n = 5, t test, the value with * are significantly different as compared to control (positive or negative) as evaluated by student’s t test (P \ 0.05)

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Evaluation of analgesic, anti-inflammatory, and neuropharmacological activities

Anti-inflammatory activity studies The Formaldehyde induced oedema model was used in the anti-inflammatory studies and the intensity and duration dependant results are summarized in Table 3. The results showed that the effects of higher dose (0.2 mg) was significantly higher as compared to lower dose (0.1 mg) of compound 1. The percent inhibition was significantly higher after 30 (*2.89-fold), 1st hour (*1.5-fold), 2nd and 3rd hour (*1.4-fold), 4th hour (*0.8-fold) of compound 1 application. Neuropharmacological activity studies Gross behaviour activity The animals were given 0.2 mg/kg of compound 1 intraperitoneally and the features observed in this gross behavioral study are given in Table 4. Effect on locomotor activity The results of the effect of compound 1 on locomotor activity in mice are given in Table 5.

Discussion The abdominal constrictions (writhes) in mice were induced by a 0.6 % acetic acid solution, when administered orally. The resulting writhes were counted for three consecutive phases, within each 20 min period up to 60 min, as the writhing response is sometimes different for various drugs (Collier et al. 1968).

Table 4 Gross behavior activity of compound 1 Sample

Motor activity

Effect on eyes

Grip strength

Urination/ diuresis

Compound 1 (0.2 mg i.p)

Decrease

Small

Weak

Increase

The test compound, nepethalate B (1) was also administered orally (300 mg/kg), and the notable differences were shown in the writhing frequency between the tested groups. It showed significant inhibition of writhes as compared to the negative control. The percentage inhibition was measured after injecting compound 1 intraperitoneally at doses of 0.1, 0.2 and 0.4 mg/kg of body weight and were found to be 53.3, 58.0 and 86.7 % of phase I respectively. These were significant (P \ 0.05) when compared with the negative control. Moreover, the percentage inhibitions of all three phases obtained for 0.1, 0.2 and 0.4 mg/kg were higher than the value obtained for Aspirin, which was used as positive control (Table 1). The acetic acid induced abdominal constriction method is widely used for the evaluation of peripheral antinociceptive activity (Khairnar et al. 2010). The acetic acid induced writhing is related to the increase in the peritoneal fluid levels of PGE2 and PGE2a (Khairnar et al. 2010). It is therefore possible that compound 1 exerts an analgesic effect probably by inhibiting synthesis or action of prostaglandins. However, the exact mechanism has not been investigated here. The formalin induced pain model is very useful for elucidating mechanism of pain and analgesia (Tjolsen et al. 1992). It is reported that formalin induces persistent pain in mice paw involving a neurogenic early phase pain, followed by an inflammatory late phase pain (Dubuisson and Dennis 1977; Tjolsen et al. 1992), which is accompanied by the release of inflammatory mediators (Hunskar et al. 1985; Murray et al. 1988). The early phase is probably a direct result of stimulation of nociceptors in the paw, while the late phase may reflect the inflammation process, and at least to some degree, the sensitization of central nociceptive neurons (Coderre et al. 1990; Coderre and Melzack 1992). Drugs that mainly act centrally such as narcotics inhibit both phases of formalin-induced pain while peripherally acting drugs inhibit only the second phase (Chen et al. 1995). The formalin induced pain test was used to find out the site of analgesic activity observed in compound 1. The test compound 1 exerted significant dose dependent inhibition of both the neurogenic first phase and inflammatory second

Table 5 CNS depressant activity of compound 1 Sample

Cage cross 15 min

Head dip 15 min

Rearing 15 min

Open field 15 min

Traction (s)

Mobility time (6 min)

Control saline ip

26 ± 2.01

28 ± 2.07

18 ± 1.12

30 ± 1.45

06 ± 0.08

Compound 1 0.2 mg ip

10 ± 0.32*

12 ± 0.22*

02 ± 0.08

03 ± 0.02

15 ± 0.05 fall

1.02 ± 0.98

4.5 ± 0.98

Diazepam 2 mg/kg ip

02 ± 0.06

04 ± 0.03

03 ± 0.02

06 ± 0.07

18 ± 0.13 fall

1.31 ± 0.55

Values are mean ± SEM, n = 5, the value with * are significantly different as compared to control (positive or negative) as evaluated by student’s t test (P \ 0.05)

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phase of formalin induced pain test. This indicates that central and peripheral mechanisms contribute in the overall analgesic effect of the test compound, nepethalate B (1). The formaldehyde induced oedema model was used in the present antiinflammatory studies. This is a process that liberates variety of mediators in two stages; the first being the release of serotonin and histamine while the second is mediated by prostaglandins. The cyclooxygenase products and the continuity between the stages are provided by kinins (Jimoha et al. 2011). The results of our studies showed a statistically significant reduction in the inflammatory growth in the hind paw of the rats, and there is a correlation between reduction in oedema and the concentration of the drug which indicates that the activity of the extract is a dose dependent. Interestingly compound 1 at both doses showed more potent anti-inflammatory effects in terms of intensity and duration as compared to standard drug aspirin. The neuropharmacological study of compound 1 was carried out on the basis of monitoring the gross behavior and the locomotory and muscle relaxation ability of the animals. The other features observed were those of frequent excretion of urine (Table 4). The results of the present study revealed that compound 1 exhibited CNS depression and reduced the locomotor activity in the mice.

Conclusion The results of the present study demonstrate that the test compound, nepethalate B (1), isolated from N. clarkei possesses significant analgesic, anti-inflammatory and CNS depressant activities. Interestingly nepethalate B (1) showed stronger analgesic and anti-inflammatory activities than the standard drug. This is the first report of a phthalate derivative which showed all these three activities. Furthermore, these results also validate the traditional use of the plant as anti-inflammatory and in some conditions associated with pain and swelling (Cigremis et al. 2010). Thus it could be possible that the traditionallyknown anti-inflammatory activity of the N. Clarkei is well associated with compound 1. Considering the results obtained in the present study, it is also important to emphasize that further biological studies on pharmacological grounds are needed to get a broader sense of the mechanism of action of analgesic, anti-inflammatory, and CNS depressant action of nepethalate B (1) and other related phthalate derivatives. Acknowledgments The authors wish to thank the Higher Education Commission (HEC), Government of Pakistan, for providing financial

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support for the current study under the National Research Program for Universities (NRPU). Conflict of interest interest.

The authors declare that there is no conflict of

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In vivo evaluation of analgesic, anti-inflammatory, and neuropharmacological activities of the chemical constituent from Nepeta clarkei.

The plant species of genus Nepeta are used to treat various human diseases and for ornamental purposes as well. Nepethalate B (1) was isolated as a re...
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