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Bioactivity-guided fractionation for analgesic properties and constituents of Vitex negundo L. seeds.


This study was undertaken to ascertain the analgesic properties of Vitex negundo L. seeds and to isolate and characterize the active constituents. Among the 80% ethanol extract and some fractions with different polarity, the acetoacetate fraction showed the highest anti-nociceptive activity in acetic acid-induced writhing test in ICR mice. The analgesic bioguided isolation of the acetoacetate fraction yielded two major lignans: 6-hydroxy-4-(4-hydroxy-3-methoxy-phenyl)-3-hydroxymethyl-7-methoxy-3, 4-dihydro-2-naphthaldehyde (1) and vitedoamine A (2). Given orally, compound (1), which was more productive, produced significant inhibitions on chemical nociception induced by intraperitoneal acetic acid and subplantar formalin injections and exhibited notable anti-inflammatory activities in dimethyl benzene-induced ear edema test in a dose-dependent manner. Since co-administration of naloxone fails to antagonize the analgesic activity of compound (1) in the formalin test, we suggest that compound (1) possesses potent analgesic effects which are most likely to be mediated by its anti-inflammatory activity rather than through opioid receptor system and therefore could partially explain the anti-nociceptive effect of V. negundo L. seeds.

[C] 2008 Elsevier GmbH. All rights reserved.

Keywords: Vitex negundo; Seeds; Analgesic; Anti-inflammatory; Bioassay-guided


Vitex negundo (VN) Linn, belonging to the genus Vitex (Verbenaceae), is a small aromatic plant with typical five foliolate leave pattern that flourishes abundantly in wastelands and widely distributed in tropical to temperate regions, native of South Asia, China, Indonesia and the Philippines, up to an altitude of 1500 m. Flavonoids, lignans, terpenoids, iridoid glycosides and alkaloids are the major classes of compounds isolated from this plant according to the former phytochemical studies on V. negundo (Diaz et al., 2003; Ono et al., 2004; Chawla et al., 1992a, b; Srinivas and Raju, 2002).

All parts of VN have been commonly used as folk medicine. The extracts of VN leaves possessed antiinflammatory, analgesic and hypouricaemic activity as well as its anti-hyperglycemic activity (Dharmasiri et al., 2003; Umamaheswari et al., 2007; Villasenor and Lamadrid, 2006). Anti-oxidant and antiandrogenic properties were reported from the flavonoid-rich fraction of the seeds (Zheng et al., 1999; Bhargava, 1989; Das et al., 2004). VN also found use for CNS depressant, anti-histamine release and hepato-preventive purposes (Gupta et al., 1999; Nair et al., 1994; Avadhoot and Rana, 1991).

Previous studies have already demonstrated the significant analgesic activity of aqueous extract from VN seeds (Zhong et al., 1996), consistent with its traditional use in southern China for the treatment of various pain disorders, such as stomachache, hernia ache, dysmenorrhea, arthralgia and piles. Based on previous investigations, we studied the analgesic activity of the aqueous ethanol extract of Vitex negundo seeds (EVNS) and the anti-nociceptive activity of the petroleum ether, dichloromethane, acetoacetate and n-butanol fractions from the aqueous ethanol extract, and found that the acetoacetate fraction had a powerful analgesic activity in preliminary experiment. The present study was designed to further examine the analgesic activity and the possible mechanism of the acetoacetate fraction as well as elucidating its chemical components and providing scientific basis for the clinical use of Vitex negundo seeds.

Materials and methods

Drugs and chemicals

The following reagents and drugs were used: EtOH (AR), petroleum ether (AR), dichloromethane (AR), ethyl acetate (AR), w-butanol (AR), formalin (AR), dimethyl benzene (AR) and acetic acid (AR) (Sinopharm chemical reagent Co. Ltd., China), morphine hydrochloride, indomethecin, dexamethasone, naloxone hydrochloride (Chengdu Pharmaceutical Factory, Chengdu, China).

Morphine hydrochloride, indomethecin, dexamethasone and naloxone hydrochloride were dissolved in physiological saline (0.9% NaCl). The vehicles used alone had no effects on the nociceptive responses in mice.

Plant material

The seeds of Vitex negundo L. (Chinese name "Huang-Jing-zi"), which was identified by Prof. Han-Chen Zheng, School of Pharmacy, Second Military Medical University, were obtained from the Wanglang National Nature Reserve, Sichuan province, in October 2006. A voucher specimen (#168) has been deposited in the herbarium of the Department of Pharmacognosy, School of Pharmacy, Second Military Medical University.


Experimental groups consisted of 10 ICR mice (18-22g) per group. They were housed at 21 [+ or -] 1[degrees]C under a 12 h light/12 h dark cycle and had free access to standard pellet diet (Purina chow) and tap water. The animals were deprived of food for 15 h before the experiment, with free access to drinking water. Each animal was used only once in the experiment. The experimental protocols were approved by the Animal Care and Use Committee of our institute and complied with the recommendations of International Association for the study of pain (Zimmermann, 1983).

Preparation of the ethanol extract and the fractions

Dried seeds of V. negundo (25 kg) were ground and extracted with 80% aqueous ethanol at room temperature. The solvent was evaporated under vacuum to afford 1600 g crude extract (yield, 6.4%). This extract (EVNS) was then suspended in water and partitioned successively with petroleum ether, dichloromethane, ethyl acetate and aqua-saturated w-butanol. Each fraction was evaporated in vacuo to yield the residues of petroleum ether 250 g (15.6%), dichloromethane 345 g (21.6%), ethyl acetate 120 g (7.5%), n-butanol 360 g (22.5%) and aqueous 526.5 g (32.9%). For pharmacological studies, EVNS and the fractions were suspended in a 1 % aqueous solution of Tween-80. The doses employed are expressed as mg of the dried extract per kg body weight except for positive drugs and compound 1.


In both of the nocifensive tests, conscious (un-anesthetized) mice were used. The doses of the positive drugs were determined on the basis of the principle of their pharmacokinetics and clinical use. The test samples were administered orally. The doses selection for the test samples was based on the results of preliminary experiments. Control groups were treated with a similar volume of vehicle that had been used to dilute the fraction.

Abdominal constriction induced by acetic acid

In the acetic acid-induced writhing test (Garcia et al., 2004), groups of overnight fasted mice (n = 10) were treated with the test samples, vehicle or indomethecin (IND), 1 h before the administration of acetic acid (0.7%, 10 ml/kg, i.p.). The number of writhing was counted for each animal, starting 3 min after acetic acid injection over the period of 12 min.

Formalin test

In the formalin test (Santos and Calixto, 1997), groups of mice were treated as above with the acetoacetate fraction and compound 1 (at the most active dose in the writhing test) or vehicle and after 60 min, each mouse was given 20 [micro]l of 5% formalin (in 0.9% saline, subplantar) into the right hind-paw. The duration of paw licking (s) as an index of painful response was determined at 0-5 min (early phase, neurogenic) and 20-25 min (late phase, inflammatory) after formalin injection. Morphine was used as a positive control drug, which was administrated at the dose of l0 mg/kg, s.c, 30 min before the test. In order to verify the possible mechanism of the acetoacetate fraction and compound 1, anti-nociception animal groups pretreated with naloxone were used. Naloxone was administered 15 min before the acetoacetate fraction, compound 1 or morphine.

Assay of dimethyl benzene-induced inflammation in mice

The test samples, vehicle or dexamethasone was administered orally 1 h separately before each dimethyl benzene topical application to the right ear. The edema was measured 1 h after dimethyl benzene treatment, and the method is the same as reported in the literature (Wei et al., 2004). The ear swelling was measured by subtracting the weight of the left ear from that of the right. The inhibitory ratio (IR) was calculated as follows: IR = (A-B) x 100/A, where edema A is edema induced by dimethyl benzene alone, and edema B is edema induced by dimethyl benzene plus sample. Each value was the mean of individual determinations in 10 mice.

Statistical analysis

All data were expressed as the mean [+ or -] S.E.M. Data weresubjectedtoANOVA followedbyDunnett's multiple comparison test. p values less than 0.05 were considered to be significant.

Results and discussion

Chemical analysis

From the acetoacetate fraction, two phenylnaphthalene-type lignans (Fig. 1) were obtained and identified as 6-hydroxy-4-(4-hydroxy-3-methoxy-phenyl)-3-hydroxy-methyl-7-methoxy-3, 4-dihydro-2-naphthaldehyde (1) and vitedoamine A (2), both of which have been previously reported and isolated from the seeds of Vitex negundo (Chawla et al., 1992b; Ono et al., 2004). The structures were elucidated unambiguously by spectroscopic methods including 1D and 2D NMR analysis and also by comparing experimental data with literature data.


Compound 1: Yellowish powder (1500 mg); 1H and 13C NMR (Chawla et al., 1992b); EIMS m/z 356 [[M] .sup.+]; HREIMS: m/z 356.1270 (calcd. for [C.sub.20][H.sub.20][O.sub.6], 356.1259).

Compound 2: White powder (80 mg); 1H and 13C NMR (Ono et al., 2004); EIMS m/z 351 [[M].sup.+]; HREIMS: m/z 351.1070 (calcd. for [C.sub.20][H.sub.17][NO.sub.5], 351.1106).

Acetic acid-induced writhing test

The results of the acetic acid-induced writhing responses in mice, which indicate the analgesic activity of the test samples, are presented in Table 1. It was found that all the extracts and fractions except for the aqueous fraction at the dose 5.0 x [10.sup.3]mg/kg body wt. caused a significant inhibition of the writhing responses induced by acetic acid as compared to the control, with values ranging from 29.52% to 55.72% protection. At 2.5 x [10.sup.3]mg/kg body wt., EVNS and the acetoacetate fraction still showed significant inhibition of the writhing responses, with inhibitions of 28.30% and 39.62%, respectively. In contrast, at a dose of 1.2 x [10.sup.3]mg/kg body wt., only the acetoacetate fraction showed any significant analgesic effect. Interestingly, compound 1 also produced significant inhibition of writhing in a dose-dependent manner at the tested doses of 2.5, 5.0 and l0 mg/kg, with inhibitions of 22.04%, 29.70% and 42.26%, respectively. These results taken together indicate that EVNS possesses strong analgesic activity, with the acetoacetate fraction possessing the highest analgesic activity as compared to other fractions. Compound 1 was found to contribute to the analgesic activity of the acetoacetate fraction. The present data give pharmacological support to the validity of the analgesic effect of Vitex negundo seeds.

Table 1. Effects of Vitex negundo L. seeds on acetic acid-induced
writhing responses in mice.

     Treatment       Dose (mg/kg     Number of writhings,    Inhibition
                      body wt.)      mean [+ or -] S.E.M.        (%)

Control          -                  38.62 [+ or -] 4.47

EVNS              5.0 x [10.sup.3]  21.75 [+ or -] 2.44 (b)     43.68
                  2.5 x [10.sup.3]  27.69 [+ or -] 1.98 (a)     28.30
                  1.2 x [10.sup.3]  32.11 [+ or -] 3.39         16.86

Petroleum ether   5.0 x [10.sup.3]  24.50 [+ or -] 3.01 (a)     36.56
fraction          2.5 x [10.sup.3]  28.49 [+ or -] 3.86         26.23
                  1.2 x [10.sup.3]  34.38 [+ or -] 4.25         10.98

Dichloromethane   5.0 x [10.sup.3]  22.82 [+ or -] 2.71 (b)     40.91
fraction          2.5 x [10.sup.3]  35.41 [+ or -] 3.26          8.31
                  1.2 x [10.sup.3]  37.53 [+ or -] 5.19          2.85

Acetoacetate      5.0 x [10.sup.3]  17.10 [+ or -] 2.91 (b)     55.72
fraction          2.5 x [10.sup.3]  23.32 [+ or -] 1.93 (b)     39.62
                  1.2 x [10.sup.3]  24.21 [+ or -] 3.09 (a)     37.31

n-Butanol         5.0 x [10.sup.3]  27.22 [+ or -] 2.18 (a)     29.52
fraction          2.5 x [10.sup.3]  33.62 [+ or -] 4.29         12.95
                  1.2 x [10.sup.3]  31.26 [+ or -] 3.07         11.06

Aqueous           5.0 x [10.sup.3]  29.47 [+ or -] 2.76         23.69
fraction          2.5 x [10.sup.3]  36.82 [+ or -] 4.22          4.66
                  1.2 x [10.sup.3]  35.13 [+ or -] 3.14          9.04

Compound (1)     10.0               22.30 [+ or -] 3.9l (a)     42.26
                  5.0               27.15 [+ or -] 1.82 (a)     29.70
                  2.5               30.11 [+ or -] 2.89         22.04

Indomethacin     10.0               16.00 [+ or -] 2.46 (b)     58.57

(a) p < 0.05.
(b) p < 0.01 compared with control.

Formalin test

In the formalin test, the vehicle-treated animals showed the mean licking times (s) of 46.63 [+ or -] 6.64 in the first phase and 28.13 [+ or -] 4.03 in the second phase (Fig. 2). Pretreatment with the acetoacetate fraction caused significant diminutions of both the first-phase (21.83 [+ or -] 2.56s) and the second-phase (5.33 [+ or -] 1.35 s) pain responses, at the tested doses of 5.0 x [10.sup.3] mg/kg. Compound 1 suppressed mainly in the later phase (first phase, 31.83 [+ or -] 3.87 s, and second phase, 9.17 [+ or -] 2.17 s). Morphine (l0 mg/kg), the reference drug, also significantly suppressed the formalin response in both phases (first phase, 3.67 [+ or -] 0.62 s, and second phase, 0.33 [+ or -] 0.16 s). When used alone, naloxone (1 mg/kg, s.c.) failed to modify the formalin-induced nociceptive responses in a significant manner (Fig. 2) (naloxone: first phase, 47.66 [+ or -] 7.81 s, and second phase, 26.76 [+ or -] 4.25 s). In the combination studies, naloxone notably antagonized the analgesic action of morphine but failed to reverse the anti-nociception produced by the acetoacetate fraction and compound 1 (Fig. 2).


Dimethyl benzene-induced ear edema test

The percent inhibition edema of EVNS was significant (p < 0.05) at the dose tested (31.54% at 5.0 x [10.sup.3] mg/kg), in comparison with the control. The acetoacetate fraction exhibited significant anti-inflammatory activity between 1.2, 2.5 and 5.0 x [10.sup.3] mg/kg, in a dose-dependent manner. Consistently, the dose-response effect was also observed for compound 1 (Table 2). At 5 mg/kg dose, the anti-inflammatory activity of compound 1 was comparable to that of dexamethasone at a dose of 1 mg/kg (54.36% vs. 45.97%). In contrast, the petroleum ether and dichloromethane fraction showed a mild anti-edema effect at the 5.0 x [10.sup.3] mg/kg dose, while the n-butanol and aqueous fraction showed fewer or no effects at this dose (Table 2). As a positive control, dexamethasone (1 mg/kg) significantly inhibited the ear edema by 45.97%. The results demonstrate the anti-inflammatory properties of Vitex negundo seeds and may justify the use of this plant for the treatment of inflammatory diseases in Chinese, folk and herbal medicine.

Table 2. Effects of Vitex negundo L. seeds on ear edema induced by
dimethyl benzene in mice.

   Treatment            Dose        Weight of edema (mg),   Inhibition
                 (mg/kg body wt.)   mean [+ or -] S.E.M.        (%)

Control          -                  2.98 [+ or -] 0.28

EVNS              5.0 x [10.sup.3]  2.04 [+ or -] 0.20 (a)     31.54

Petroleum ether   5.0 x [10.sup.3]  1.81 [+ or -] 0.23 (a)     39.26

Dichloromethane   5.0 x [10.sup.3]  2.03 [+ or -] 0.19 (a)     31.55

Acetoacetate      5.0 x [10.sup.3]  1.16 [+ or -] 0.14 (b)     61.07
fraction          2.5 x [10.sup.3]  1.53 [+ or -] 0.18 (b)     48.66
                  1.2 x [10.sup.3]  1.98 [+ or -] 0.21 (a)     33.56

n-Butanol         5.0 x [10.sup.3]  2.56 [+ or -] 0.30         14.09

Aqueous           5.0 x [10.sup.3]  2.77 [+ or -] 0.25          7.05

Compound (1)     10.0               1.06 [+ or -] 0.09 (b)     64.42
                  5.0               1.36 [+ or -] 0.l0 (b)     54.36
                  2.5               2.48 [+ or -] 0.22         16.78

Dexamethasone     1.0               1.61 [+ or -] 0.23 (b)     45.97

(a) p < 0.05.
(b) p < 0.01 compared with control.


Natural products exhibiting analgesic properties are of great interest for a variety of reasons. The seed of Vitex negundo L. is a rich source of several biological molecules, such as organic molecules, produced via the secondary metabolism, including terpenoids, lignans, flavones and alkaloids. It has been suggested that the aqueous extract of Vitex negundo seeds showed anti-nociceptive activity (Zhong et al., 1996). In the present work, we have fractionated the seed of Vitex negundo and found the main analgesic fraction. From this effective fraction, we have obtained an interesting phenyldihydronaphthalene-type lignan, compound 1, which exhibited significant anti-nociceptive activity at tested doses and contributed to the analgesic properties of Vitex negundo seeds extract.

The acetic acid-induced writhing method was widely used for the evaluation of peripheral anti-nociceptive activity, which was able to determine the anti-nociceptive effect of compounds or dose levels that might appear inactive in other methods like tail-flick test (Bentley et al., 1983). However, it was known that constriction induced by acetic acid was considered to be a non-selective anti-nociceptive model, since acetic acid indirectly induced the release of endogenous mediators and stimulated the nociceptive neurons that were sensitive to non-steroidal anti-inflammatory drugs (NSAIDs) (Sanchez-Mateo et al., 2006). Our results indicated that the acetoacetate fraction and compound 1 of Vitex negundo seeds could significantly reduce the number of writhing on animal model, showing powerful anti-nociceptive effects. However, the results of this writhing test alone did not ascertain whether the anti-nociceptive effect was central or peripheral.

In order to confirm it, the formalin test was carried out. The advantage of the formalin model of nociception was that it could discriminate pain in its central and/or peripheral components. The test consists of two different phases that can be separated in time: the first one is generated in the periphery through the activation of nociceptive neurons by the direct action of formalin and the second phase occurs through the activation of the ventral horn neurons at the spinal chord level (Tjolsen et al., 1992). Central analgesic drugs, such as narcotics, inhibited equally in both phases, while peripherally acting drugs, such as steroids (hydrocortisone, dexamethasone) and NSAIDs (aspirin), suppressed mainly in the later phase (Trongsakul et al., 2003). In this test, the acetoacetate fraction of Vitex negundo seeds at more effective doses in the writhing test could reduce the duration of the paw licking (s) obviously in both the first phase (neurogenic) and the second phase (inflammatory) of the formalin test. It was reasonable that the acetoacetate fraction of Vitex negundo seeds had the same anti-nociceptive activity as central analgesic drugs, while compound 1 acted peripherally and suppressed mainly in the second phase similar to those classical anti-inflammatory drugs. The observed central anti-nociceptive activity of the acetoacetate fraction reported in this study could be due to the presence of other active constituents which were not isolated. Further bioassay-guided isolation of compounds with central analgesic activity is currently being conducted in our laboratory.

To verify possible anti-nociceptive mechanisms, we had examined the effect of naloxone, a non-selected opioid receptor antagonist, on the anti-nociceptive activity of the acetoacetate fraction and compound 1. Since naloxone failed to antagonize the anti-nociception produced by the acetoacetate fraction and compound 1, it was thought that the observed analgesic effect is not mediated through opioid receptors.

As we know, prostaglandins play an important role in pain progress in chemical nociception models (Deraedt et al., 1980; Santos et al., 1998) and are the target of action of commonly used anti-inflammatory drugs. Several other inflammatory mediators, such as sympathomimetic amines, tumor necrosis factor-[alpha], interleukin-1[beta] and interleukin-8, are also involved in the nociceptive response to chemical stimulus in mice (Ferreira et al., 1988, 1993; Duarte et al., 1988; Santos et al., 1998; Ribeiro et al., 2000). Therefore, dimethyl benzene-induced ear edema test was further employed to evaluate the anti-inflammatory activity of the acetoacetate fraction and compound 1. Both of the test samples showed significant anti-inflammatory activity, consistent with the indications revealed in the second phase (inflammatory) of the formalin test, implying that the analgesic activity of the acetoacetate fraction and compound 1 may be mediated by its antiinflammatory action.

In conclusion, the study demonstrated the analgesic activity of the acetoacetate fraction of Vitex negundo seeds in the test models of nociception induced by chemical stimuli. Bioassay-guided isolation from the acetoacetate fraction led to a productive compound, 6-hydroxy-4-(4-hydroxy-3-methoxy-phenyl)-3-hydroxy-methyl-7-methoxy-3, 4-dihydro-2-naphthaldehyde, with potent analgesic properties that could partially explain the analgesic effect of V. negundo L. seeds extract. It is suggested that the observed anti-nociceptive activity might be related to its anti-inflammatory activity, which merited further studies regarding the precise site and the mechanism of action.


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C.-J. Zheng (a), (1), W.-Z. Tang (b), (1), B.-K. Huang (a), T. Han (a), Q.-Y. Zhang (a), H. Zhang (a), L.-P. Qin (a), *

(a) Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, PR China

(b) Department of Stomatology, Changhai Hospital, Second Military Medical University, 174 Changhai Road, Shanghai 200433, PR China

* Corresponding author. Tel./fax: +8621 25070394.

E-mail address: (L.-P. Qin).

(1) C.-J. Zheng and W. -Z. Tang have contributed equally to this work.

doi: 10.1016/j.phymed.2008.12.001

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Author:Zheng, C.-J.; Tang, W.-Z.; Huang, B.-K.; Han, T.; Zhang, Q.-Y.; Zhang, H.; Qin, L.-P.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Jun 1, 2009
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