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Antinociceptive Activity of the Crude Methanolic Extract and Different Fractions of Viola serpens Wall.

Byline: Rukhsana Ghaffar, Muhammad Saeed, Manzoor Ahmad, Haroon Khan, Omer Shehzad and Attiqa Naz

Summary: The purpose of our study was to evaluate the toxicity and antinociceptive effect of the crude extract/fractions of Viola serpens in animal studies. The extract was found absolutely safe up to the doses of 2 g/kg in acute toxicity test. When studied in acetic acid induced writhing test, significant (p<0.05) attenuation of painful sensation at doses, 100, 200 and 300 mg/kg (i.p.) was caused by the extract. Upon fractionation, hexane fraction was the most prominent fraction in pain reduction followed by chloroform and ethyl acetate fractions in a dose-dependent manner. Similarly, in formalin induced tail flick test, the extract antagonized the nociception in both phases, being more pronounced in late phase 100, 200 and 300 mg/kg (i.p). The n-hexane was the most active fraction in pain reduction followed by chloroform and ethyl acetate fractions.

The crude extract and fractions of V. serpens possessed marked antinociceptive effects and seemed to be a safe drug that could provide a scientific rationale for its folk uses as analgesic.

Keywords: Viola serpens, Antinociceptive, Acetic-induced writhing test, Formalin test, Acute toxicity


Plants being a regular source of natural medicinal constituents (natural) are use for the management of therapeutic disorders since old civilizations [1]. In modern medicines, naturally isolated constituents are mostly used throughout the world now-a-days and in industrialized countries approximately half of these have been adopted as useful therapeutic drugs [2]. According to the World Health Organization report approximately 70-80 % population of the world still relies on the traditional medicine for cure of various diseases [3-5]. Medicinal plants are gateways for opening new doors [6]. Natural products provided significant and useful therapeutic management to various ailments which synthetic product may not be effective due to their side effects [7, 8].

Nociception being non-specific undesirable feelings caused mostly due to harmful (external/ internal) stimuli. Analgesics are pain relieving drugs either acting directly or following the mechanism peripheral pain [9]. Ayureda, an ancient therapeutic system has proved much helpful in developing new useful drugs [10].

Viola serpens Wall belongs to the family Violaceae which consists of about twenty three genera and 930 species [11]. There are total 500 reported species of viola, out of which 17 are found in Pakistan [12]. Viola serpens commonly known as Gul-e-banafsha [13] is found mostly in mountains at an elevation of around 800-3000 m [14]. Various species of the genus Viola are distributed in India, Afghanistan, Bhutan, Malaysia, Indonesia, Thailand, Sri Lanka, China, Myanmar, Nepal, Kashmir and Pakistan [15]. In folk medicine it is used as an antipyretic, laxative, emollient, expectorant, purgative, anti-asthmatic, anti-cancer, against jaundice, hepatitis, skin diseases and for management of constipation [16-18]. It is also used for treatment of headache, cough, cold, and dermatitis, diseases of kidneys, liver, lungs and urinary infections [19-21]. The phytochemical investigations showed that V. serpens contains flavonoids, glycosides, alkaloids, tannins and coumarins [22].

It also contains methyl salicylate, mucilage, sugar, violin gum and saponin [17, 18]. Its antioxidant constituents are ascorbic acid, peroxidase, ascorbate oxidase and catalase [23]. This study was planned for evaluating the acute toxicity and antinociceptive effects of crude extract and fractions of V. serpens in animal models.


Plant Collection

The plant collection was done from District Shangla (Village, Puran), Khyber Pukhtunkhwa, Pakistan, in the month of April, 2011. Plant specimen was identified by Dr. Mohmmad Ibrar, (Taxonomist) professor in the Department of Botany, University of Peshawar. The plant specimen was deposited in the department's herbarium with voucher # Bot.20158 (PUP). The whole plant collected was weighted before and after drying at ambient temperature.

Extraction and Fractionation

The shade dried powdered plant (10 kg) was soaked in 25 L methanol for 10 days at room temperature with vigorous stirring on daily basis. Filtrate was obtained by using colorless filter cloth followed by Whatmann filter paper. Filtrates were dried with rotary evaporator (R-210, Buchi, Switzerland) at 40-45AdegC and fitted with a re-circulating chiller (NESLAB instruments). Crude methanolic extract was obtained from the procedure (1.57 kg) which was treated with various solvents (based on polarity) for fractionation. A 5L separating funnel was used for fractionation of the crude methanolic extract of the plant (1.32 kg). 1L distilled water mixed with 1.5 L n-hexane was added in separating funnel (shacked vigorously) and allowed to stand till the separation of immiscible. n-Hexane (upper layer) was collected from the separating funnel and dried (706 g).

The n-hexane fraction followed the fractions of chloroform, ethyl acetate, n-butanol and aqueous with dried masses 17 g, 22.7 g, 35 g and 45 g respectively obtained in the same manner.

Experimental Animals

BALB/C mice of both sexes, male and female of weights 20-25 g purchased from the animal house of NIH (National Institute of Health Islamabad). They were bred in the animal house, Department of Pharmacy, University of Malakand. These animals were kept at standard environment and maintained at parameters of 25AdegC temperature and 12/12 h light/dark cycle with free access to food and water (standard laboratory formula). Rules of the ethical committee were followed before and after the experiments. Food and health, guidelines of the mice were adjusted throughout the experiments according to the rules provided by the institute of laboratory animal resources, Commission on life sciences, National Research Council.

Acute Toxicity

The crude methanolic extract of the whole plant at three different doses ranging from 1g/kg to 2 g/kg were used for determination of the acute toxicity. The mice were uniformly grouped into three, comprising six mice each. The negative control group was treated with distilled water (10 ml/kg) whereas the rest of the two groups were treated with the crude methanolic extract (1 g/kg and 2 g/kg). The animals were closely observed in the first 6 hrs of the test doses administration and continued for 24 hrs. During this period no mortality was found. The animals were kept in the animal house for two weeks with regular observations on the alternate days. The animals were found remain healthy and active in this period [24].

Acetic Acid Induced Writhing Reflex

The animals were arranged into seventeen groups of, six mice each. Group I (negative control) and II (positive control) were treated with normal saline 10 ml/kg and diclofenac sodium (10 mg/kg) respectively. 1% acetic acid at a dose of 10 ml/kg body weight was used for pain induction. Animals recommended guidelines were followed for feeding, but supply of food was ceased before two hours of the start of the activity [25]. Administration of the different fractions (n-hexane, Chloroform, Ethyl acetate, and aqueous) and the crude methanolic extract were done to the groups-III to XVII at the doses of 100, 200 and 300 mg/kg. All groups were administered with 1% acetic acid (0.01 ml i.p) after 30 min of the above mentioned treatments. The abdominal writhes (constrictions), after 5 minutes of the acetic acid injection (0.01 ml), were counted for 10 min [25]. The analgesic effect was calculated in percentage as per the following formula:

% of Analgesic Effect = 100 - No of writhes in the test animals / No of writhes in control animal x 100

Formalin Test

The formalin test was conducted on the bases of previous study [26]. BALB/C mice of either sex were selected in18-22 gm body weights. The animals were divided into seventeen groups, each having six animals. Pains were induced by injecting 0.05 ml of 2.5% formalin (40% formaldehyde) in saline to the right paw (hind) of the animal. The group I (control) and the group II (standard) received normal saline and Diclofenac sodium in the dose (10 mg/kg, i.p) respectively. Rest of the groups (III - XVII) were treated with the respective fraction (n-hexane, Chloroform, Ethyl acetate, and aqueous) or crude methanolic extract at three different doses (100, 200 and 300 mg/kg), 30 min. before the formalin injection. The time spent in licking and flicking the injected paw was recorded and as an indicator of pain response. Measurement of the responses was done during the first 5 min. (early phase) and from 20 to 30 min. (late phase) after formalin injection.

Result and Discussion

V. serpens Wall crude extract and the subsequent fractions, n-hexane, chloroform, ethyl acetate and aqueous at different doses 1000 and 2000 mg/kg, i.p were tested for acute toxicity. After the 24 hours close observations and complete two weeks survey no mortality was found in the test animals. The animals remain active and alive even after the high doses administration of the plant doses, proved to be a safe herbal drug even at high doses. The plant can be safely recommended for different pharmacological uses.

Effect of Crude Extract/Fractions of V. serpens Wall. in Acetic Acid Induced Writhing Test

The results of crude extract/fraction of V. serpens in acetic acid induced writhing test at various doses (100, 200 and 300 mg/kg i.p.) are shown in Table-1. Diclofenac was used as a standard drug. The crude extract and all the fractions, except aqueous fraction showed significant effects in acetic acid induced writhing test in a dose dependent manner. The crude extract showed more significant effect followed by n-hexane, chloroform and ethyl acetate with significant values of 19.77, 21, 31.50 and 34.74 along with percent inhibition values 70.05%, 68.8% 50.37 % 50.37 % at a dose of 300 mg/kg body weight respectively. Whereas, the aqueous fraction does not produce any significant anti-nociceptive effect at test doses.

Table-1: Effects of the crude methanolic extract of V. serpens and its fraction in acetic acid induced in mice.

Drugs###Dose mg/kg###No. of writhing (10min)###% Protection

Saline###10 ml/kg###66+-2.90











Ethyl acetate###200###37.12+-2.75*###43.76






Effect of Crude Extract/Fractions of V. serpens in Formalin Induced Nociception Test

The formalin induced nociception test results in both phases are shown in the Table-2 In the initial phase (0-5 min) the crude extract showed dose dependent anti-nociceptive effect with maximum pain reduction (35%) while 64.8% was observed in the second phase (15-30 min) i.p. at a dose of 300 mg/kg body weight. Crude extract was subjected to fractions, marked changes were observed from the fractions. The fraction of n-hexane showed a dose dependent pain subsiding effect followed by chloroform, ethyl acetate and aqueous fractions. In both the early and 38.23 %, 26.5%, 25.0 and 23.0% respectively. In the late phase the values of percent inhibition were 55.21 %, 37.14%, 38.57% and 25% respectively.

Table-2: Effects on the crude methanolic extract of V. serpens and its fractions in formalin induced pain in mice.

Drugs###Dose mg/kg###Early phase (0-5min)###Late phase (15-30 min)

Saline###10 ml/kg###68+-1.79###93+-2.30











Ethyl acetate###200###56+-2.50###36+-2.35**






The current study revealed marked antinociceptive effect of crude extract/fractions of V. serpens in chemically induced protocols of acetic acid induced writhing and formalin induced tests.

Acetic acid induced abdominal constriction/writhes is an assay used for the measurement/ determination of anti-nociceptive activity by the peripheral mechanism [27, 28]. As a result of acetic acid administration, release of pain mediators causes the increased lipoxygenase and prostaglandin (PGE2, PGE2[alpha]) production in the peritoneal fluid [25, 29]. Increase in the capillary permeability ultimately caused inflammatory pain stimulation through the peritoneal receptors [30].

The reductions in writhes clearly indicate the anti-nociceptive activity. The plant extract and its different fractions produced more pronounced anti-nociceptive effects. The effect increased in a dose dependent in each fraction whereas, decreased with the polarity. The crude extract showed more attenuated analgesic effect followed by the n-hexane, ethyl acetate, chloroform and aqueous fractions. In the three test doses, 300 mg/kg (i.p) was more effective as compared with the low doses (100 and 200 mg/kg, i.p). The plant followed a peripheral pathway which may be due to the inhibition of the local peritoneal receptors that may be responsible along with certain other mediators for reduction in the release of cyclooxygenase or lipoxygenase enzymes.

Acetic acid induced writhing test being nonspecific, formalin induced paw licking test was used for mechanistic approach. It is a qualitative measurement of centrally acting analgesia [31, 32]. Formalin induced nociception being a biphasic analgesic behavioral protocol with involve two clearly different stimuli. The chemical released in the early phase (neurogenic phase) are the bradykinin and substance P. The late inflammatory phase, involve the release of prostaglandins, histamine, serotonin and bradykinin [32] The plant extract/fractions of V. serpens showed more pronounced anti-nociceptive effect in late phase as compared to the early phase. The effect of the plant being more significant in the second phase clearly indicates its similarity with the non-steroidal anti-inflammatory drug like indomethacine and aspirin [33, 35]. Centrally acting dugs like narcotic analgesics show effectiveness in both the early and late phases [33, 35].

The main constituents in the plant of V. serpens are alkaloids, saponins, tannins and flavonoids which may be responsible for inhibiting the mediators release like prostaglandins, histamine, serotonin or bradykinin as pain suppressors in the late phase [24, 36] The outcome of the study is that the anti-nociceptive property of the plant was mediated through the peripheral mechanism; augmented by interference of centrally acting pain mediators. Due to the pronounced anti-nociceptive effect, the plant may be effectively recommended for clinical purpose.


It is concluded that the crude extract and subsequent fractions of V. serpens showed marked antinociceptive activity. The peripheral pain mediators in-line with the central mediators, partly explained the possible underline mechanisms. Thus, the study provides a scientific rationale for its folk uses as analgesic. Moreover, further study on the phytochemical parameters regarding the isolation of secondary metabolites would provide clear picture of our findings.


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Author:Ghaffar, Rukhsana; Saeed, Muhammad; Ahmad, Manzoor; Khan, Haroon; Shehzad, Omer; Naz, Attiqa
Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Geographic Code:9PAKI
Date:Aug 31, 2017
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