Research Article

Analgesic activity of Lawsonia inermis leaves extract in swiss albino mice

Manivannan R.*, Aeganathan R.

Department of Chemistry, Government Arts College (Autonomous), Kumbakonam, Tamilnadu-612 001, India

*For correspondence

Dr. Manivannan R.,

Department of Chemistry, Government Arts College (Autonomous), Kumbakonam, Tamilnadu, India.

Email: manickam_mani@yaho







Received: 14 April 2016

Accepted: 23 May 2016


Objective: Lawsonia inermis an important traditional medicinal plant used in various ailments and the indigenous plant as a medicament for treatment of various inflammatory conditions is well documented in literature. The analgesic activity was evaluated by hot-plate and acetic acid induced writhing methods.

Methods: The leaves of L. inermis were collected from the local area in and around Kumbakonam, Thanjavur District (India). About 1500 g of the powder was extracted using Soxhlet apparatus for 12 h using 5.0 L of methanol as solvent. The test extracts was administered intraperitoneally at a dose of 250 and 500 mg/kg while morphine sulfate (5 mg/kg) and diclofenac sodium (100 mg/kg) served as standards.

Results: All the extract displayed significant analgesic effect (P<0.05-0.001) in acetic acid and heat induced pain models in a dose dependent manner.

Conclusions: The results suggest that all the extracts of L. inermis possesses potent analgesic properties, which support its use in traditional medicine and suggesting that the plant should be further investigated for its pharmacological active natural products.

Keywords: Analgesic activity, Hot-plate, Acetic acid induced writhing method, Lawsonia inermis


In recent years there has been a phenomenal rise in the interest of scientific community to explore the pharmacological actions of herbs or to confirm the claims made about them in the official books of Ayurveda.1 Henna is a finely ground brown or green powder originating from dried leaves of the plant Lawsonia inermis Linn, which is grown in dry tropical and subtropical zones, including North Africa, India, Sri Lanka, and the Middle East.2 It is used as medicinal plant because of its attributed antibacterial, antifungal, antiamoebiasis, astringent, antihemorrhagic, hypotensive and sedative effect.3 L. inermis have carbohydrates, proteins, flavonoids, tannins, as well as phenolic compounds, alkaloids, terpenoids, quinones, coumarins, xanthones, and fatty acids. This plant has been used for the treatment of epilepsy and jaundice, for dyeing gray hair, and for malignant ulcers.4 Moreover, the leaves have a bitter bad taste with vulnerary and diuretic effects which are useful in the treatment of headache, hemicranias, lumbago, bronchitis, boils, ophthalmia, syphilitics, sores, amenorrhea, scabies, and diseases of the spleen and favor the growth of the hair.5 Analgesics are drugs that selectively relieve pain by acting in the CNS or on peripheral pain mechanisms, without significantly altering consciousness. Analgesics are divided into two groups, opioid analgesic and non-opioid analgesic.6 In most instances, these analgesic drugs, particularly opioid and nonsteroidal anti-inflammatory drugs (NSAIDs), can only relieve 50% of the pain in about 30% of patients.7 Studies have shown that opiates cause physical dependency, tolerance, and addiction while NSAIDs usually cause gastrointestinal disorders.8 As such, research to discover other alternatives to treat pain is crucial. Over 50% of all modern drugs are natural product origin and they play an important role in drug development programs of the pharmaceutical industry.9 Many of these herbs with analgesic activity had been used without any adverse effects. The present study aimed to evaluate the analgesic activity of methanol petroleum and ethyl acetate extract of L. inermis in animal models.

Materials and Methods

Plant material

The leaves of L. inermis were collected from the local area in and around Kumbakonam, Thanjavur District (India) in the month of January – February and authenticated by Dr. N. Ramakrishnan, Head & Associate Professor, Department of Botany, Government Arts College (Autonomous), Kumbakonam, Tamilnadu, India with voucher specimen number GACBOT 501 was deposited at the Herbarium of the Department of Botany, in our institution. After authentication, the leaves were collected in bulk quantity, washed under running tap water to remove the adhering dirt and shade dried at room temperature.


The leaves of L. inermis were washed with distilled water to remove dirt. The leaves were further air dried under shade and made into fine powder by using hand homogenizer and sieved through sieve No.40 and the fine powder was for extraction procedure and evaluation. About 1500 g of the powder was extracted using Soxhlet apparatus for 12 h using 5.0 L of methanol as solvent. The extract was made free from solvent by keeping it on water bath at 50-60°C for about 6 h and gave a yield of 16.35%. The methanol crude extract was further partitioned using first with petroleum ether and then successively extracted with ethyl acetate. These extracts were concentrated under reduced temperature and pressure on a rotary evaporator. The yields of the pet-ether and ethyl acetate extracts were 5.6 and 9.8%, respectively.

Test animals

Young Swiss Albino mice of either sex, aged 4 - 5 weeks and average weight of 20 - 25 g were procured from the animal house, Government Arts College (Autonomous), Bharathidasan University, Kumbakonam, Tamilnadu, India and used for the pharmacological investigations. The animals were kept in polypropylene cages under standard laboratory condition maintained at room temperature of 25 ± 2°C, relative humidity of 56-60%, and proper dark–light cycle. All animals were fed with the standard rodent pellet diet, and water adlibitum. Before starting the experiment on animals, the experimental protocol was subjected to the scrutiny of the Institutional Animal Ethics Committee (IAEC), Bharathidasan University, Trichirappalli, Tamilnadu, India (Approval No. BDU/IAEC/2011/31/29.03.2011).

Toxicity testing

The LD50 was determined using the graphical method10 in mice. Mice were divided into control and test groups (n=6). The test groups received the extract per orally at the doses of 500 -2000 mg/kg were administered i.p. Control group received 1% DMSO (10 ml/kg i.p.). Animals were critically observed for possible behavioral changes, allergic reactions and mortality at least once during the first 30 minutes of dosing followed by occasional observation for first 24 h and continued for 72 h for the recording. Confirmatory test was carried out and the LD50 was calculated from the graph of percent mortality against profit log dose of the extract.

Analgesic activity

Hot plate method (thermal stimulation)

Evaluation of analgesic activity of the plant extract was also carried out using hot plate method11. Experimental animals of either sex were randomly selected and divided into eight groups consisting of six mice in each group for control, standard and test groups respectively. Control group was treated with 1% DMSO at the dose of 10 ml/kg body weight; test groups were treated at dose of 250 and 500 mg/kg; standard were treated with morphine (5 mg/kg) administered intraperitoneal (i.p.) route. All animals were lowered onto the surface of a hot plate (55 ± 0.5°C) enclosed with cylindrical glass and the time for the animal to jump or lick the fore limb was noted as the reaction time. A cut off period of 30 seconds was observed to avoid damage to the paw. The observations were made before and after administration of respective drugs at 30 min, 60 min, and at the end of 90 min. Reaction time of the test and standard groups were compared with the control.

Acetic acid induced writhing test (chemical stimulation)

The analgesic activity of the samples was evaluated using acetic acid induced writhing method12; albino mice (30 - 40 g) were divided into eight groups each consisting of six animals. Control group served as (1% DMSO) at the dose of 10 ml/kg body weight. Second group served as standard (received diclofenac sodium 100 mg/kg), while third and fourth groups received plant extract were treated at doses of 250 and 500 mg/kg orally. In this method, acetic acid is administered intra-peritoneal to the experimental animals to create pain sensation. Writhing in animals was produced by i.p. administration of 300 mg/kg acetic acid (3%) solution. The writhing movements were observed and counted for 30 min after acetic acid administration. The number of writhes of test groups at different dose levels, and standard were compared with the control. The percent inhibition of writhing count of the treated group was calculated from the mean writhing count of the control group.

Percentage inhibition was calculated using the following formula:

% inhibition= { (Wc - Wt) × 100 } / Wc

Where, Wc = No. of writhes in control group, Wt = No. of writhes in test group

Statistical analysis

The experimental results were expressed in multiple comparisons of Mean ± SEM and was carried out by one-way analysis of variance (ANOVA) followed by Dunnet Multiple Comparisons Test and statistical significance was defined as P <0.05.

Results and Discussion

It's a long and tedious process to isolate pure, pharmacologically active constitutes from plants. Thus, it is necessary to have methods available which eliminate unnecessary separation procedures. Our previous study described the isolation and structure elucidations of five known flavonoids such as apigenin, 5, 7, 4¢-trihydroxy-6, 3¢,5¢-trimethoxyflavone, pecto-linarigenin, apigenin-7-O-β-D-glucoside, pecto-linarin obtained for the first time from the leaves of L. inermis.13

Pain is a condition which is regularly dealt with in daily clinical practice. Hence, any attempt to contribute an easily available analgesic drug from the available flora is always accepted without any reluctance. A thermal nociception model such as hot plate test was used to evaluate central analgesic activity. Hot-plate test is a widely used model for neurologic pain, and centrally acting analgesic agents can increase reaction time in hot-plate test through their action at the spinal cord level.14 The different plant extracts, when given in doses of 250 and 500 mg/kg, i.p. elicited a significant analgesic activity in the hot plate as evidenced by increase in latency time in seconds (Table 1) as compared with vehicle control. The increase in latency time was dose dependent. Latency time was noted at 0, 30, 60 and 90 min after the administration of vehicle, standard and plant extracts. The results of the hot plat test revealed that the latency time was significantly (P<0.05) increased from 9.36 ± 1.15 to 14.10 ± 1.13 at the dose of 250 to 500 mg/kg. The effect was dose dependent and the maximum effect was observed at 90 min as shown in Table 1. The most significant (P<0.01) increase in latency time noticed against 500 mg/kg of L. inermis was 14.10 ± 1.13 whereas; the percent inhibition of the standard morphine sulphate analgesic was 15.78 at 90 min. The result of hot plate test indicates that the extract has marked analgesic effects with a reasonable safety profile and possesses the ability to reduce centrally mediated pain. This attempt is to prove the efficacy of the plant extract as a potential analgesic drug and to demonstrate a positive result.

Table 1: Effect of L. inermis leaves extracts in hot-plate test in mice.



Reaction time (in sec) (M±SD)

0 min

30 min

60 min

90 min


Control (1% DMSO)

06.06 ± 0.67

06.17 ± 0.68

06.37 ± 0.64

06.56 ± 0.66


Standard (Morphine Sulphate + 5 mg/kg)

06.10 ± 0.94

08.96 ± 0.99

14.22 ± 1.12

15.78 ± 1.13


MeOH extract 250 mg/kg 500 mg/kg

06.20 ± 1.05 06.24 ± 1.04

07.12 ± 1.11 07.86 ± 1.10

8.83 ± 1.14 9.67 ± 1.13

09.36 ± 1.15 10.58 ± 1.12


Ether extract 250 mg/kg 500 mg/kg

06.26 ± 1.02 06.25 ± 1.05

07.88 ± 1.15 08.27 ± 1.14

9.11 ± 1.13 10.19 ± 1.12

10.91 ± 1.16 12.13 ± 1.12


EtOAc extract 250 mg/kg 500 mg/kg

06.23 ± 1.10 06.18 ± 1.00

08.14 ± 1.12 08.67 ± 1.10

10.23 ± 1.13 12.82 ± 1.13

10.34 ± 1.14 14.10 ± 1.13

Data expressed as Mean ± SEM, n = 6 in each group done by one way ANOVA followed by Dunnet test.

Table 2: Effect of L. inermis leaves extracts on acetic acid induced writhing in mice.

Treatment (n =6)

Dose (mg/kg)

No. of writhes (Per 30 Min)

% inhibition

Control (1% DMSO)

10 mL/Kg

39.14 ± 1.26


Diclofenac sodium

50 mg/kg

06.92 ± 1.07


MeOH extract

250 mg/kg

500 mg/kg

16.24 ± 1.22

13.20 ± 1.11



Ether extract

250 mg/kg

500 mg/kg

13.02 ± 1.10

10.82 ± 1.06



EtOAc extract

250 mg/kg

500 mg/kg

10.22 ± 1.09

07.88 ± 1.08



Data expressed as Mean ± SEM, n = 6 in each group by one way ANOVA followed by Dunnet test.

The analgesic activity was also observed in the acetic acid - induced pain model. Acetic acid-induced writhing is a well recommended protocol in evaluating medicinal agents for their analgesic property. As shown in Table 2, indicate that there is a significant reduction in writhing in mice treated with the extract of L. inermis and standard in comparison with the control group. The significant pain reduction of both the plant extracts with two selected doses i.e. 250 and 500 mg/kg might be due to the presence of analgesic principles acting with the prostaglandin pathways in writhing method in mice at different time intervals. In methanol, petroleum ether and ethyl acetate extracts (500 mg/kg) reduced writhing count significantly, inhibiting pain by 66.27, 72.35 and 79.87% compared to control whereas diclofenac sodium showed 82.31% inhibition at the dose of 100 mg/kg as compared to the control, and the results were statistically significant. The substance inhibiting the writhing will have analgesic effect preferably by inhibition of prostaglandin synthesis, a peripheral mechanism of pain inhibition.15 Peritoneal administration of acetic acid (0.7%) induces endogenous pain mediators, such as prostaglandins, histamine, serotonin (5-HT), bradykinin and substance P that sensitize pain nerve endings.16 The released prostaglandins, mainly prostacyclin (PGI2) and to lesser extent PGE2 and PGF have been held responsible for pain sensation.17 These findings strongly recommend that L. inermis has peripheral analgesic activity and their mechanisms of action may be mediated through inhibition of local peritoneal receptors which may be the involvement of cyclooxygenase inhibition potential. Many peripherally or centrally acting analgesics have been isolated from plants and thus require extensive studies to explore more analgesic agents from natural sources.18 A number of alkaloids, flavonoids, steroids, and tannin isolated from medicinal plants have been reported to possess significant analgesic activity.19 The flavonoids (apigenin, 5, 7, 4

¢-trihydroxy-6, 3¢, 5¢-trimethoxyflavone, pectolinarigenin, apigenin-7-O-β-D-glucoside, pectolinarin), which are presence to these extracts, might contribute to the analgesic activity observed.


Present study revealed both peripheral and central analgesic activities of L. inermis leaves extract established in vivo models, which strongly supports its use in traditional medicine. This attempt is to prove the efficacy of the L. inermis leaves extract as a potential analgesic drug and to demonstrate a positive result. These extracts or its isolated chemical components could represent a new resource for the development of new plant-based therapy useful in the control of pain.

Funding: No funding sources

Conflict of interest: None declared


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