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Analgesic and anti-inflammatory activity of caryophyllene oxide from Annona squamosa L. bark.

ABSTRACT

Caryophyllene oxide was isolated from an unsaponified petroleum ether extract of the bark of Annona squamosa and studied for its analgesic and anti-inflammatory activity. Caryophyllene oxide at the doses of 12.5 and 25 mg/kg body wt. and unsaponified petroleum ether extract at a dose of 50 mg/kg body wt. showed significant central as we as peripheral analgesic, along with anti-inflammatory, activity. These activities of caryophyllene oxide were comparable with the standard drug used in the respective experiments.

[C] 2009 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords;

Annona squamosa

Unsaponified petroleum ether extract

Caryophyllene oxide

Analgesic

Anti-inflammatory

1. Introduction

Annona squamosa L. (Annonaceae) is a tree occurs wild and is also cultivated throughout India (Kirtikar and Basu 1991). Its leaves are used as insecticidal and antispasmodic agents and are used in the treatment of rheumatism and painful spleen. The plant is reported to possess analgesic, anti-inflammatory (Dash et al. 2001), antipyretic, antiulcer, and antiseptic and abortifacient activities (Akolkar et al. 1992). Its use as an insecticidal agent has been investigated by several workers (Cheema et al. 1985) and various phytochemical, pharmacological, antibacterial and antiovulatory studies have already been carried out with the seed extracts (Vohra et al. 1975). Post-cortical antifertility activity of A. squamosa has also been reported from studies with the seed extract (Mishra et al. 1979).

A tetrahydroisoquinoline alkaloid with cardiotonic activity has been isolated from the leaves of Annona squamosa (Wagner et al. 1980) along with a bioactive acetogenin from its bark (Li et al. 1990). A novel diazepine, squamolone was also isolated from this plant (Yang and Chi-Ming 1972). The purpose of the study reported here was to isolate the pure constituent responsible for analgesic and anti-inflammatory activity from the bark of the plant.

2. Materials and methods

2.1. Plant material

Bark of Annona squamosa L. was collected from Ahmednagar district, Maharashtra, in May 2006. A voucher specimen has been deposited in the herbarium of the Botanical Survey of India, Pune, under reference F. No. 148530.

2.2. Extraction and isolation

Shade-dried powdered material weighing 1 kg was extracted by soxhlet using petroleum ether (40-60 [degrees]C). The solvent was evaporated in vacuo to obtain the crude petroleum ether extract. The extract was further purified by separating saponified and unsaponified matter through treatment of alcoholic alkali (Har-borne 1998). The unsaponified petroleum ether extract (USPE) (10g) obtained was applied to a column of Silica gel 60 (60-120 mesh) packed in benzene slurry and the column was developed with benzene, from which were collected 14 fractions of 300-400 ml each. Fractions 6-10 were combined on the basis of similar TLC pattern (Si gel plates, benzene, vanilline-sulphuric acid spray). These fractions were further resolved by preparative TLC on silica gel [GF.sub.254] using benzene as a mobile phase, resulting in isolation of two minor compounds along with caryophyllene oxide (32 mg, [R sub.F]0.4). Caryophyllene oxide: white amorphous powder, m.p.: 60-61 [degrees]C. Molecular formula [C.sub.15][H.sub.24]O. The IR, .[1.sup.H] NMR, .[13.sup.C] NMR and mass spectra of the compound were matched with those reported in the literature (Ahmed et al. 2005).

2.3. Animals

All the experiments were carried out using male Swiss albino mice (20-25g each) and Wistar rats (150-200g each). The animals had free access to food and water and they were housed under a natural (12 h each) light-dark cycle with access to standard pellet chow and water ad libitum. The animals were acclimatized for at least 5 days to the laboratory conditions before performing the experiments. The experimental protocol was approved by the Institutional Animal Ethics Committee (Registration No. 372/01/a/ CPCSEA). In all experimental models, six animals were used in each group.

2.4. Analgesic activity

The central analgesic activity of the test drug was studied against thermal stimuli using the hot plate test (Eddy and Leimback 1953), while peripheral analgesic activity of the test drug was evaluated using the acetic acid-induced writhing test (Koster et al. 1959).

2.5. Eddy's hot plate test

The initial reaction times of all the animals of control and test groups were recorded by putting them on the hot plate maintained at 55[+ or -] 0.5[degrees]C. Licking of paw or jumping was taken as the index of reaction to heat. The albino mice were divided into five groups. USPE (50 mg/kg body wt.), Caryophyllene oxide (12.5 and 25 mg/kg body wt.), or pentazocin lactate injection (50 mg/kg body wt.) were administered by intra peritoneal route. The first group served as control and received vehicle only (1% dimethyl formamide in water for injection). The post-treatment reaction time of each animal was recorded at 30, 60, 90,120 and 180 min.

2.6. Acetic acid induced writhing

The albino mice were divided into five groups. USPE (50 mg/kg body wt.), Caryophyllene oxide (12.5 and 25 mg/kg body wt.) or aspirin (100 mg/kg body wt.) were administered one hour prior to intra peritoneal injection of 0.6% v/v acetic acid. Five minutes after the intra peritoneal injection of acetic acid, the number of writhings during the following 20min was counted. Control mice received vehicle only (1% dimethyl formamide in water for injection).

2.7. Anti-inflammatory activity

Albino rats of Wistar strain of either sex were divided into five groups. Acute inflammation was produced by sub plantar injection of 0.1 ml of 1% suspension of carrageenan with 2% gum acacia in normal saline, in the right hind paw of rats, one hour after oral administration of USPE (50 mg/kg body wt.), Caryophyllene oxide (12.5 and 25 mg/kg body wt.) or aspirin (100 mg/kg body wt.). Control rats were received vehicle only (1% dimethyl formamide in water for injection). The paw volume was measured plethysmometrically (Ego Basil, Italy) at 0, 1, 2, and 3 h after the carrageenan injection. The difference between '0' readings and readings after 1, 2, and 3 h, respectively, was taken as the volume of edema (Winter et al. 1962).
Table 1

Effect of Caryophyllene oxide on thermic stimulus-induced pain in
mice (hot plate test).

Treatment       Latency to lick the paws (Sec[+ or -]S.E.M.

                Pre drug reaction time   30 min

Control         12.45[+ or -]0.50        8.50[+ or -]0.92

Pentazocin (50  10.96[+ or -]0.34        8.33[+ or -]1.43
mg/kg body
wt.)

USPE (50 mg/kg   8.35 [+ or -]1.02       9.26[+ or -]0.99
body wt.)

Caryophyllene    8.92[+ or -]1.2         6.66[+ or -]0.88
oxide (12.5
mg/kg body
wt.)

Caryophyllene    9.45[+ or -]0.94    5.50[+ or -]0.76
oxide (25
mg/kg body
wt.)

Treatment

                  60 min               90 min

Control          9.00[+ or -]1.15     8.83[+ or -]0.54

Pentazocin (50   8.83[+ or -]0.87     16.16[+ or -]1.24 *
mg/kg body
wt.)

USPE (50 mg/kg    20[+ or -] 0.18 *     20[+ or -]0.94 *
body wt.)

Caryophyllene   10.33[+ or -]0.95*    12.33[+ or -]1.20 *
oxide (12.5
mg/kg body
wt.)

Caryophyllene    8.33[+ or -]0.91     12.66[+ or -]0.98 *
oxide (25
mg/kg body
wt.)

Treatment

                120 min                   180 min

Control          8.16[+ or -]1.10          8.00[ + or -]0.89

Pentazocin (50  17.50[+ or -]0.95 *        7.08[+ or -]0.58
mg/kg body
wt.)

USPE (50 mg/kg     20[+ or -]0.26 *       10.74[+ or -]0.20
body wt.)

Caryophyllene    14.5[+ or -]1.02 *        8.00[+ or -]0.68
oxide (12.5
mg/kg body
wt.)

Caryophyllene   17.00 [+ or -] 0.96 *      9.33[+ or  -]0.80
oxide (25
mg/kg body
wt.)

Values are mean [+ or -]S.E.M., n= 6,
* p<0.05, significant* compared to control.
USPE- Unsaponified Petroleum Ether Extract.


2.8. Statistical analysis

All the results were statistically analyzed by student's t-test and expressed as mean[+ or -]S.E.M. Results were regarded as significant when p<0.05.

3. Results

The effect of unsaponified petroleum ether extract and pure caryophyllene oxide were evaluated for central as well as peripheral analgesic, along with anti-inflammatory, activity. Table 1 shows the results on thermic stimulus induced pain (Eddy's hot plate test) in mice. Pretreatment with pentazocin or USPE or caryophyllene oxide did not produce any significant changes of paw licking time in the early phase of pain. However, in the late phase, a dose-dependent and significant (p<0.05) increase in licking time was observed in mice treated with caryophyllene oxide as well as with USPE and pentazocin. The maximum activity was observed with caryophyllene oxide (25 mg/kg body wt., i.p.) at the 120 min time interval, which is comparable to the standard pentazocin. The maximum analgesia induced by USPE (50 mg/kg body wt., i.p.) was at the 60 min time interval and persisted up to 120 min.

Table 2 shows the response of mice to acetic acid-induced writhing. Treatment of USPE, caryophyllene oxide and standard aspirin significantly (p<0.05) reduce the number of writhes. The inhibitions were 64.89%, 57.87% and 75.19% for USPE and two doses of pure caryophyllene oxide, respectively. At the dose of 25 mg/kg body wt., caryophyllene oxide inhibited the writhing response almost to the same degree as aspirin (74.41%). Table 3 shows the response of rats to the carrageenan induced paw edema. The results obtained with USPE, caryophyllene oxide and standard aspirin show significant (p<0.05) inhibition of inflammatory edema at the first and second hours after the carrageenan treatment. The effect of caryophyllene oxide at a dose of 25 mg/kg body wt. p.o. was comparable to standard aspirin. The dose-dependent inhibition of edema was observed with caryophyllene oxide treatment.
Table 2

Effect Caryolphyllene oxide on acetic acid-induced writhing
test in mice

Treatment                        No.                 Inhibition (%)
                                 of writhing

Control                          42.33[+ or -]1.62

Aspirin (100 mg/kg body wt.)     10.83[+ or -]0.65 *  74.41

USPE (50 mg/kg body wt.)         14.86[+ or -]1.21 *  64.89

Caryophyllene oxide              17.83[+ or -]1.10 *  57.87
(12.5 mg/kg body wt.)

Caryophyllene oxide              10.50[+ or -]1.05 *  75.19
(25 mg/kg body wt.)

Values are mean [+ or -] S.E.M., n = 6.
* p<0.05. significant * compared to control.
USPE-Unsaponified Petroleum Ether Extract.

Table 3

Effect of Caryophyllene oxide on carrageenan-induced paw edema in rats.

Treatment                                  Increase in paw volume
                                           in ml.

                                           1 Hour
Control                                    0.56[+ or -]0.036
Aspirin (100 mg/kg body wt.)               0.23[+ or -]0.017 *
USPE (50 mg/kg body wt.)                   0.26[+ or -]0.016 *
Caryophyllene oxide (12.5 mg/kg body wt.)  0.25[+ or -]0.016 *
Caryophyllene oxide (25 mg/kg body wt.)    0.23[+ or -]0.015 *

Treatment
                                           2 Hour

Control                                    0.55[+ or -]0.029
Aspirin (100 mg/kg body wt.)               0.20[+ or -]0.011 *
USPE (50 mg/kg body wt.)                   0.29[+ or -]0.024 *
Caryophyllene oxide (12.5 mg/kg body wt.)  0.30[+ or -]0.021 *
Caryophyllene oxide (25 mg/kg body wt.)    0.27[+ or -]0.013 *

Treatment
                                           3 Hour

Control                                    0.65[+ or -]0.064
Aspirin (100 mg/kg body wt.)               0.66[+ or -]0.036
USPE (50 mg/kg body wt.)                   0.61[+ or -]0.028
Caryophyllene oxide (12.5 mg/kg body wt.)  0,45[+ or -]0.028 *
Caryophyllene oxide (25 mg/kg body wt.)    0.66[+ or -]0.036

Values are mean [+ or -]S.E.M., n=6, * <0.05, significant * compared to
control.

USPE-Unsaponified Petroleum Ether Extract.


4. Discussion

The results of the present study show that the unsaponified petroleum ether extract and caryophyllene oxide isolated from this extract of Annona squamosa bark exhibit significant analgesic and anti-inflammatory activities. The results obtained in the analgesic test experiments appear to suggest that USPE and pure caryophyllene oxide isolated from this extract possess centrally and peripherally mediated analgesic properties. The central analgesic action may be mediated via inhibition of central pain receptors, while the peripheral analgesic effect may be mediated through inhibition of cyclooxygenase and/or lipoxygenase (and other inflammatory mediators). This hypothesis is in consonance with those of Eddy and Leimback (1953), Williamson et al. (1996) and Koster et al. (1959) who postulated that acetic acid writhing and hot plate test methods are useful techniques for the evaluation of centrally and peripherally acting analgesic drugs, respectively. Caryophyllene oxide produced an analgesic effect against thermal induced pain stimuli in mice at various time points post-treatment. The effect observed was dose-dependent and statistically significant. The hot plate test is considered to be selective for opioid-like compounds, which are centrally acting analgesics in several animal species (Janseen et al. 1963). Furthermore, the effect of USPE was initiated first and appears for long duration when compared with different doses of caryophyllene oxide in the hot plate test. This might be due to the other constituents present in the USPE, such as the kaurane-type diterpenes, which might contribute to the central analgesic activity (Li et al. 1990).

The carrageenan-induced rat paw edema is a suitable test for evaluating anti-inflammatory drugs that has frequently been used to assess the anti-edematous effect of natural products (Panthong et al. 2003). Development of the edema in the paw of the rat after injection of carrageenan is a biphasic event. The initial phase observed during the first hour is attributed to the release of histamine and serotonin. The second phase of edema is due to the release of prostaglandins, protease and lysosomes (Vinegar et al. 1969; Crunkhon and Meacock 1971). The USPE at 50 mg/kg body wt. and caryophyllene oxide at doses of 12.5 and 25 mg/kg body wt. shows significant activity in the first and second hours. This might be due to the inhibition of the biphasic response induced by the carrageenan.

5. Conclusion

Caryophyllene oxide administered intra peritoneally exhibits antinociceptive activity and might exerts its effect through diverse mechanisms that may involve both central and peripheral pathways. Further pharmacodyanamic investigations are required to understand the analgesic and anti-inflammatory activity exhibited by caryophyllene oxide.

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M.J. Chavan (a), P.S. Wakte (b), D.B. Shinde (b), *

(a) Department of Pharmacognosy, Amrutvahini College of Pharmacy, Sangamner S.K. 422 608, Dist-Ahmednagar, M.S.. India

(b) Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431 001, M.S., India

* Corresponding author.

E-mail address:dbshinde97@rediffmail.com (D.B. Shinde).

0944-7113/$-see front matter[C] 2009 Elsevier GmbH. All rights reserved.

doi:10.1016/j.phymed.2009.05.016
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Title Annotation:Short communication
Author:Chavan, M.J.; Wakte, P.S.; Shinde, D.B.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:1USA
Date:Feb 1, 2010
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