Bioassay-guided isolation of anti-inflammatory and antinociceptive compound from Plumbago zeylanica leaf

Abstract

Plumbago zeylanica Linn. (Plumbaginaceae) is used in the treatment of various inflammatory ailments in traditional medicines. In order to validate these ethnobotanical practices, the anti-inflammatory and antinociceptive activities of various leaf extracts (petroleum ether (60–80°), chloroform, acetone, ethanol, and aqueous) were studied using in vivo experimental models at two dose levels (200 and 400 mg/kg, p.o.). Anti-inflammatory activity was tested using the carrageenan induced rat hind paw edema method while analgesic activity was studied using the hot plate and formalin induced models. Diclofenac (100 mg/ kg) was used as the reference standard in both anti-inflammatory and analgesic models and morphine (10 mg/ kg, i.p.) was used as the reference standard in the formalin induced analgesic model. The acetone extract significantly (p < 0.01) reduced inflammation in the rats when compared to the control group. As for the analgesia effect, the acetone and petroleum ether extracts significantly (p < 0.01) decreased the pain stimulus only in the later phase of the formalin test, suggesting that the drug could be peripherally acting. Bioassay-guided fractionation of the acetone extract led to the isolation and identification of plumbagin. Structure elucidation of plumbagin confirmed it as 5-hydroxy-2-methyl-1,4-naphthoquinone, a naphthaquinone derivative, through spectral techniques.

Keywords::

• Anti-inflammatory
• analgesic
• IR spectroscopy
• plumbagin
• Plumbago zeylanica
• UV spectroscopy

Introduction

The term inflammation refers to the events that occur in the tissues in response to an invading pathogen or due to the presence of a noxious substance. It is usually associated with pain as a secondary process resulting from the release of analgesic mediators. Therapy of inflammatory diseases is usually directed at the inflammatory processes. Though many non-steroidal anti-inflammatory agents (NSAIDs) have been prepared and marketed, these drugs are known to provoke gastrointestinal irritation. Due to such reasons there is an increased demand by patients to use natural products with therapeutic activities (Osadebe & Okoye, 2003). The present observations relate to the discovery of one such herb, Plumbago zeylanica Linn. (Plumbaginaceae), commonly known as Chitrak, which has been found to have efficacy traditionally in reducing inflammation (Kritikar & Basu, 1975). It grows wildly and abundantly in India. Folk practitioners and local people of Malaya eat the leaves of this plant to cause abortion (Anonymous, 2001). The aerial parts of the plant are reported to be used in various ailments such as rheumatic pain, scabies, skin diseases, wounds, inflammation, etc. (Nguyen et al., 2004), while the roots are reported to have antioxidant (Tilak et al., 2004), central nervous system (CNS) stimulant (Bopaiah & Pradhan, 2001), antimicrobial (Ahmad et al., 1998), antiplasmodial (Simonsen et al., 1998), wound healing (Reddy et al., 2002), hypolipidemic, and antiatherosclerotic (Sharma et al., 1991) activities.

So far, during a literature survey no scientific evidence was found regarding the anti-inflammatory and analgesic activities of P. zeylanica leaf. The present study was carried out to evaluate the analgesic and anti-inflammatory claim of the local people of Orissa and to identify the bioactive constituent.

Materials and methods

Plant material

The leaves of P. zeylanica were collected in the month of March from Kanyakumari district, Tamil Nadu, and positively identified by Dr. H. S. Chatree, Botanist, Government Arts and Science College, Mandsaur, Madhya Pradesh. A voucher specimen (P/006/2006/BRNCOP) was deposited in the herbarium of the Department of Pharmacognosy, B. R. Nahata College of Pharmacy (BRNCP), Mandsaur for future reference.

Source of chemicals

All the chemicals/drugs and solvents used were of analytical grade and purchased from Merck Chemicals, Worli, Mumbai.

Preparation of extracts for preliminary activity testing

The leaves of the plant were shade-dried and powdered (25–45 mesh size). The powdered material was extracted using petroleum ether (60–80°) for 72 h and successively extracted with chloroform, acetone, ethanol, and water for 72 h each in a Soxhlet apparatus (Jarald et al., 2008). The obtained extracts were evaporated under vacuum to give residues. The percentage yields of the various extracts are given in Table 1.

Table 1.  Preliminary phytochemical studies and percentage yields of various extracts and fractions of P. zeylanica.

Plant extract	Yield (%, w/w)	Constituents
Pz.L-P	2.83	Fats, steroids, and naphthaquinone
Pz.L-C	2.73	Steroids and naphthaquinone
Pz.L-A	1.2	Tannins, flavonoids, triterpenoids, and naphthaquinone
Pz.L-E	14.26	Carbohydrates, tannins, flavonoids, and saponins
Pz.L-W	22.2	Carbohydrates, tannins, alkaloids, flavonoids, and saponins
F1	26.69	Tannins
F2	45.81	Tannins, flavonoids, and triterpenoids
F3	2.18	Plumbagin
F4	21.43	Triterpenoids

Pz.L, P. zeylanica leaves; P, petroleum ether (60–80°); C, chloroform; A, acetone; E, ethanol; W, aqueous; F₁, fraction 1; F₂, fraction 2; F₃, fraction 3; F₄, fraction 4.

Fractionation of extract using chromatographic separation and isolation of active constituents

The acetone extract (10 g) was subjected to chromatographic separation by loading onto a glass column (90 × 3 cm) using silica gel (# 60–120) as stationary phase. Gradient elution was performed using n-hexane containing increasing amounts of ethyl acetate as the solvent system. The fractions, each of 100–150 mL eluant, were collected on the basis of the color band appearing and according to the total volume (100 mL) of solvent eluted. The column was eluted till 90% of the extract loaded eluted out. Twenty-three fractions were collected and fractions showing similar thin layer chromatography (TLC) pattern were pooled together. Finally, four fractions were obtained (F₁–F₄). The percentage yields of fractions were determined with respect to the total weight of the acetone extract taken (Table 1).

Fractions eluted showing only one spot (17, 18, and 19) on TLC (n-hexane:ethyl formate, 9:1; R_f 0.37) were pooled and evaporated to dryness, yielding plumbagin (F₃). The melting point of the isolated plumbagin was recorded. For TLC experiments, precoated plates (silica gel 60) were used. The purity of the isolated compound was assessed by recording the ultraviolet (UV) and infrared (IR) [Shimadzu IR spectrophotometer (8400S)] spectra of the compound (Nayana et al., 2005).

Preliminary phytochemical screening of extracts and fractions

In order to determine the presence of alkaloids, glycosides, flavones, tannins, terpenes, sterols, saponins, fats, and sugars, a preliminary phytochemical study with plant extracts and fractions was performed (Khandelwal, 2005).

Animals and treatment

After obtaining approval from the Institutional Animal Ethics Committee (registration number 918/ac/05/CPCSEA), Wistar rats weighing 150–180 g and adult albino mice (18–25 g) of either sex were procured from the animal house of BRNCP, Mandsaur and used for investigation. The animals were housed in standard environmental conditions of temperature (21 ± 2°C), humidity (55 ± 10%), and 12 h light–dark cycle. Rats were supplied with a standard pellet diet and water ad libitum. The extracts and fractions that were not soluble in water were suspended in 1% Tween 80 just before administration.

Acute toxicity studies

Acute toxicity tests of the extracts and fractions were carried out according to Organisation for Economic Co-operation and Development (OECD) guideline no. 420 (OECD, 2001). Female Wistar rats (150–180 g) were used for this study. After a sighting study, the starting dose of 2000 mg/kg (p.o.) of test samples was given to various extract groups containing five animals in each group. The treated animals were monitored for 14 days for mortality and general behavior. The extracts were found to be safe up to the dose of 2000 mg/kg and, from the results, two different doses of 200 and 400 mg/kg were chosen for further experimentation. Fractions F₁, F₂, and F₄ were also safe up to 2000 mg/kg, so 400 mg/ kg was chosen for further experimentation, while F₃ was safe only up to 10 mg/kg, so a dose level of 2 mg/kg was selected.

Preparation of test samples for bioassays

All extracts were administered at 200 and 400 mg/kg doses after adding to a 1% Tween 80 suspension in distilled H₂O. F₁, F₂, and F₄ were studied at 200 mg/kg and F₃ at 2 mg/kg. The control group of animals received the same experimental handling as those of the test groups except that the drug treatment was replaced with appropriate volumes of the dosing vehicle. Either diclofenac (100 mg/kg) or morphine 10 mg/kg (i.p.) in 1% Tween 80 was used as the reference drug according to the animal model.

Anti-inflammatory activity

The carrageenan induced hind paw edema model was used in the study. Rat right hind paw edema was induced by subplantar injection of 0.1 mL of 1% (w/v) carrageenan suspension in normal saline 1 h after oral administration of the extract/fraction/drug (Moody et al., 2006). The animals were divided into various groups of six each, fasted for 6 h, and deprived of water only during the experiment. The deprivation of water was to ensure uniform hydration and to minimize variability in edematous response (Winter et al., 1963). The control groups were treated with 0.2 mL of Tween 80 (negative control) or 100 mg/kg of diclofenac (positive control) (Ojewole, 2006). Immediately after injection of the phlogistic agent, volume readings (Vt) were obtained for each rat at 0, 60, 120, 180, and 240 min, with the aid of a plethysmometer. Edema was expressed as an increase in volume of the paw, and the percentage of inhibition for each rat and each group was obtained as follows:

Percentage of inhibition = (Vt – Vo) control – (Vt – Vo) treated/(Vt – Vo) control × 100

Analgesic activity in hot plate model

The hot plate test was conducted according to the procedure described by Gupta et al. (2005). Mice that reacted within 15 s and that did not show a large variation when tested on four separate occasions were selected for the studies. Sixteen groups (n = 6) of mice were selected for the present study. Group one served as negative control (received 1% Tween 80 solution) and group two received the standard drug, diclofenac, at a dose of 100 mg/kg. The remaining groups received the various extracts and fractions. Mice were screened by placing them on a hot plate maintained at 55 ± 1°C, and the reaction time in seconds for licking of the hind paw or jumping was recorded.

Analgesic activity using formalin induced model

Adult albino mice (18–25 g) of either sex were divided into groups of six mice each and were pre-treated as follows: group I, vehicle (negative control); group II, received the standard drug diclofenac (100 mg/kg) in 1% Tween 80; group III received the standard drug morphine 10 mg/kg (i.p.); the remaining groups received various extracts and fractions. Thirty minutes after this treatment, 20 µL of 2.5% formalin in saline was injected subcutaneously to the hind paws of the mice. The total time spent licking the injected paw was recorded. The data are expressed as total licking time in the early phase (0–5 min) and the later phase (15–30 min) after formalin injection (Younga et al., 2005).

Statistical analysis

All values are presented as mean ± SEM. Differences between means were assessed by one-way analysis of variance (ANOVA), followed by Dunnett’s test; p < 0.05 was considered significant.

Results

Preliminary phytochemical screening

Phytochemicals present and percentage yields of the various extracts and fractions of P. zeylanica are given in Table 1.

Effect of extracts and fractions in carrageenan induced inflammation

Injection of the phlogistic agent, carrageenan, in the control group caused localized edema starting at 0.05 h after the injection. The swelling increased progressively to a maximum volume of 1.13 ± 0.03 mL at 3 h after the carrageenan injection. The acetone extract of P. zeylanica leaves showed significant anti-inflammatory activity, and the activity of the acetone extract was equally pronounced as that of the reference drug, diclofenac (Table 2). The acetone extract of the leaves from bioassay-guided fractionation gave four main fractions (F₁–F₄) as shown in Table 1, which led to the isolation of plumbagin (F₃). Rats pre-treated with F₂ and F₃ showed more prominent activity compared with the other two fractions in both bioassay models (Table 3). This behavior was similar to that with the standard drug diclofenac.

Table 2.  Anti-inflammatory activity of various extracts of P. zeylanica.

Treatment	Dose (mg/kg)	Average volume of mercury displaced (mL)
		0 h	1 h	2 h	3 h	4 h	5 h
Control	–	0.64 ± 0.02	0.93 ± 0.02	0.90 ± 0.00	1.13 ± 0.03	1.08 ± 0.04	0.93 ± 0.04
Diclofenac	100	0.56 ± 0.03	0.82 ± 0.04 (11.8)	0.68 ± 0.00* (24.4)	0.79 ± 0.00** (30.3)	0.67 ± 0.02** (37.9)	0.56 ± 0.06** (39.78)
Pz.L-P	200	0.56 ± 0.03	0.84 ± 0.03 (9.6)	0.82 ± 0.03 (8.8)	0.90 ± 0.02 (20)	0.92 ± 0.03 (14.8)	0.86 ± 0.03 (7.5)
	400	0.56 ± 0.02	0.82 ± 0.07 (11.8)	0.83 ± 0.03 (7.7)	0.78 ± 0.05** (30.9)	0.91 ± 0.05 (15.7)	0.71 ± 0.02* (23.6)
Pz.L-C	200	0.54 ± 0.02	0.83 ± 0.01 (10.7)	0.84 ± 0.05 (6.6)	0.97 ± 0.01 (14.1)	0.93 ± 0.02 (13.8)	0.80 ± 0.02 (13.9)
	400	0.53 ± 0.01	0.84 ± 0.05 (9.6)	0.80 ± 0.04 (11.1)	0.96 ± 0.04 (15)	0.92 ± 0.02 (14.8)	0.74 ± 0.07 (20.4)
Pz.L-A	200	0.58 ± 0.04	0.78 ± 0.02 (16.1)	0.79 ± 0.04 (12.2)	1.00 ± 0.01* (11.5)	0.90 ± 0.02*(16.6)	0.79 ± 0.01 (23.6)
	400	0.60 ± 0.03	0.76 ± 0.01 (18.2)	0.74 ± 0.02* (17.2)	0.74 ± 0.05** (34.51)	0.70 ± 0.04** (35.18)	0.60 ± 0.01** (35.48)
Pz.L-E	200	0.54 ± 0.03	0.84 ± 0.03 (9.6)	0.81 ± 0.03 (10)	0.96 ± 0.03 (15)	0.94 ± 0.02 (12.9)	0.93 ± 0.04 (0)
	400	0.58 ± 0.00	0.88 ± 0.03 (5.3)	0.82 ± 0.03 (8.8)	0.98 ± 0.06 (13.2)	0.90 ± 0.06 (16.6)	0.78 ± 0.04 (16.1)
Pz.L-W	200	0.54 ± 0.02	0.84 ± 0.03 (9.6)	0.81 ± 0.05 (10)	0.98 ± 0.04 (13.2)	0.99 ± 0.00 (8.3)	0.86 ± 0.03 (7.5)
	400	0.56 ± 0.01	0.80 ± 0.01 (13.9)	0.80 ± 0.03 (11.1)	0.90 ± 0.04 (20)	0.93 ± 0.02 (13.8)	0.77 ± 0.05 (17.2)

Each value represents the mean ± SEM (n = 6); *p < 0.05, **p < 0.01 vs. control (Dunnett’s test). Values in parenthesis represents percentage inhibition.

Table 3.  Anti-inflammatory activity of various fractions of acetone extract of P. zeylanica leaf.

Treatment	Dose (mg/kg)	Average volume of mercury displaced (mL)
		0 h	1 h	2 h	3 h	4 h	5 h
Control		0.60 ± 0.02	0.74 ± 0.04	0.92 ± 0.02	1.04 ± 0.03	1.12 ± 0.03	1.05 ± 0.03
Diclofenac	100	0.57 ± 0.02	0.66 ± 0.03 (10.81)	0.69 ± 0.01** (25)	0.73 ± 0.03** (28.80)	0.77 ± 0.01** (31.25)	0.64 ± 0.02** (39.04)
F1	200	0.56 ± 0.01	0.63 ± 0.01 (14.86)	0.98 ± 0.02 (-6.52)	0.91 ± 0.05 (12.5)	1.02 ± 0.01 (8.92)	0.99 ± 0.06 (5.71)
F2	200	0.56 ± 0.01	0.65 ± 0.02 (12.16)	0.80 ± 0.04* (13.04)	0.88 ± 0.06* (15.38)	0.96 ± 0.06* (14.28)	0.87 ± 0.07* (17.14)
F3	2	0.55 ± 0.02	0.63 ± 0.01(14.86)	0.73 0.03** (20.65)	0.70 ± 0.04** (32.69)	0.79 ± 0.02** (29.46)	0.67 ± 0.01** (36.19)
F4	200	0.62 ± 0.01	0.68 ± 0.01 (8.10)	0.90 ± 0.06 (2.17)	0.89 ± 0.03 (14.42)	0.99 ± 0.06 (11.60)	0.96 ± 0.06 (8.57)

Each value represents the mean ± SEM (n = 6); *p < 0.05, **p < 0.01 vs. control (Dunnett’s test). Values in parenthesis represents percentage inhibition.

Effect of extracts and fractions on hot plate reaction time in mice

As shown in Table 4, the extracts of the leaves did not show any significant analgesic activity at both dose levels, and among the fractions only F₃ showed a potent antinociceptive activity against hot plate reaction time (35.13, 161.5, 258.92, 181.33% inhibition, respectively) (Table 5).

Table 4.  Effect of various extracts of P. zeylanica in mice on hot plate reaction time.

Treatment	Dose (mg/kg)	15 min	30 min	60 min	120 min
Control	2 ml/kg	4.83 ± 0.47	5.33 ± 0.56	5.50 ± 0.56	5.50 ± 0.62
Diclofenac	100	5.42 ± 0.47 (12.21)	8.58 ± 0.42** (60.97)	14.56 ± 1.20** (164.7)	20.99 ± 0.97** (281.63)
Pz.L-P	200	4.33 ± 0.88 (−10.35)	5.83 ± 0.47 (9.38)	7.33 ± 0.66 (33.27)	6.00 ± 0.36 (24.18)
	400	4.16 ± 0.83 (−13.87)	5.33 ± 0.33 (0)	6.83 ± 0.74 (24.18)	5.50 ± 0.22 (0)
Pz.L-C	200	4.33 ± 0.80 (−10.35)	5.16 ± 0.47 (3.01)	6.67 ± 0.84 (21.27)	6.67 ± 0.21 (21.27)
	400	4.00 ± 0.44 (−17.18)	5.00 ± 0.73 (6.19)	6.16 ± 1.07 (12.00)	6.50 ± 0.67 (18.18)
Pz.L-A	200	4.50 ± 0.67 (6.83)	4.50 ± 0.42 (−15.57)	5.22 ± 0.18 (−5.09)	5.83 ± 0.16 (5.66)
	400	5.00 ± 0.36 (3.51)	4.83 ± 0.37 (9.38)	6.67 ± 0.84 (21.27)	6.16 ± 0.83 (10.71)
Pz.L-E	200	4.16 ± 0.47 (−13.87)	4.80 ± 0.42 (9.94)	5.05 ± 0.45 (−8.08)	5.83 ± 0.79 (5.66)
	400	5.66 ± 0.21 (−17.18)	5.00 ± 0.36 (6.19)	7.00 ± 0.68 (27.27)	5.66 ± 0.49 (2.90)
Pz.L-W	200	4.16 ± 0.70 (−13.87)	5.83 ± 0.30 (9.38)	5.66 ± 0.75 (2.90)	6.83 ± 0.47 (19.47)
	400	5.00 ± 0.73 (3.51)	4.66 ± 0.33 (−12.57)	6.83 ± 0.91 (24.18)	5.66 ± 0.49 (2.90)

Each value represents the mean ± SEM (n = 6); **p < 0.01 vs. control (Dunnett’s test).

Table 5.  Effect of various fractions of acetone extract of P. zeylanica leaf on hot plate reaction time in mice.

Treatment	Dose (mg/kg)	15 min	30 min	60 min	120 min
Control	–	6.66 ± 0.95	6.50 ± 1.25	6.50 ± 1.08	6.16 ± 1.13
Diclofenac	100	8.16 ± 1.04 (22.52)	8.16 ± 1.27 (25.53)	16.16 ± 1.90^** (148.6)	23.16 ± 1.16** (275.97)
F1	200	4.98 ± 0.83 (−25.22)	6.00 ± 1.54 (−7.69)	7.66 ± 1.30 (17.84)	4.66 ± 1.83 (−24.35)
F2	200	7.33 ± 1.05 (10.06)	6.50 ± 1.36 (0)	4.33 ± 0.84 (−33.38)	7.00 ± 1.12 (−13.63)
F3	2	9.00 ± 0.82* (35.13)	17.00 ± 2.30** (161.5)	23.33 ± 1.76** (258.92)	17.33 ± 1.42** (181.33)
F4	200	5.48 ± 0.82 (−17.77)	5.33 ± 1.04 (−18)	5.00 ± 0.68 (−23.07)	7.50 ± 1.66 (−8.08)

Each value represents the mean ± SEM (n = 6); *p < 0.05, **p < 0.01 vs. control (Dunnett’s test).

Effect of extracts and fractions on formalin induced nociception in mice

The first (0–5 min) and second (15–30 min) phases of the formalin test correspond to neurogenic and anti-inflammatory pain, respectively. Though the petroleum ether and acetone extracts had analgesic effect only in the later phase, the petroleum ether extract showed a weaker activity in this assay model. Neurogenic induced pain was significantly blocked only by F₃, and not by the other extracts at both dose levels, whereas the petroleum ether and acetone extracts significantly blocked pain emitting from inflammation along with F₂ and F₃. The extracts were found to inhibit the pain resulting from inflammation better than the neurogenic induced pain. The standard drug diclofenac was also active at the later phase (Table 6). The lower dose of acetone extract was found to be more active. Among the isolated fractions, F₂ and F₃ showed a potent antinociceptive activity against formalin induced nociception (65.44 and 73.44% inhibition, respectively) with significance of F₃ in inhibiting pain in both phases (Table 7). The other fractions (F₁, F₄) were ineffective against all assay models.

Table 6.  Effect of P. zeylanica extracts on formalin induced nociception in mice.

Treatment	Dose (mg/kg)	Duration of pain response (s)		Inhibition (%)
		First phase (0–5 min)	Second phase (15–30 min)	First phase	Second phase
Control	–	62.66 ± 2.98	177.83 ± 4.35	–	–
Diclofenac	100	61.66 ± 3.19	100.67 ± 6.76**	1.59	43.38
Morphine	10	30.89 ± 2.56**	40.86 ± 5.72**	50.7	77.02
Pz.L-P	200	94.83 ± 3.06	152.68 ± 4.87*	–51.34	14.14
	400	87.50 ± 8.74	141.00 ± 5.77**	–39.64	20.71
Pz.L-C	200	91.00 ± 7.16	171.67 ± 7.49	–45.22	3.46
	400	71.16 ± 6.6	149.33 ± 8.19	–13.56	16.02
Pz.L-A	200	58. 50 ± 4.78	30.16 ± 3.70**	6.63	83.03
	400	60.83 ± 2.41	30.66 ± 5.31**	2.92	82.75
Pz.L-E	200	70.66 ± 8.72	177.33 ± 4.33	–11.71	0.28
	400	75.66 ± 5.19	163.67 ± 6.46	–20.74	7.96
Pz.L-W	200	73.16 ± 6.93	180.80 ± 5.95	–16.75	–1.67
	400	68.00 ± 3.17	170.17 ± 2.16	–8.52	4.3

Each value represents the mean ± SEM (n = 6); *p < 0.05, **p < 0.01 vs. control (Dunnett’s test).

Table 7.  Effect of P. zeylanica fractions on formalin induced nociception in mice.

Treatment	Dose (mg/kg)	Duration of pain response (s)		Inhibition (%)
		First phase (0–5 min)	Second phase (15–30 min)	First phase	Second phase
Control	–	54.00 ± 4.78	177.00 ± 6.70	–	–
Diclofenac	100	47.16 ± 5.61	39.33 ± 4.63	12.66	77.77
Morphine	10	30.00 ± 3.40**	41.66 ± 2.70**	44.44	76.46
F1	200	54.33 ± 4.71	169.17 ± 6.96	0.60	4.44
F2	200	57.66 ± 6.83	61.16 ± 3.46**	6.77	65.44
F3	2	39.44 ± 4.63**	47.16 ± 5.54**	26.96	73.44
F4	200	53.50 ± 2.96	170.83 ± 3.50	0.74	3.48

Each value represents the mean ± SEM (n = 6); **p < 0.01 vs. control (Dunnett’s test).

Plumbagin (F₃)

The melting point of isolated plumbagin (F₃) was recorded to be 78°C, which matched data given in the literature. Isolated plumbagin was dissolved in acetone and spotted on precoated plates of silica gel. Among the different solvent systems employed in the mobile phase, n-hexane:ethyl formate (9:1) resolved plumbagin at R_f 0.37. The spot of plumbagin showed a yellow coloration in daylight, a faint quenching in UV (long wave), and a magenta pink color with 10% alcoholic KOH solution. The purity of the compound was further confirmed by recording UV and IR spectra of the isolated compound.

UV (methanol) λ_max 265 nm (UV1-Thermospectronic spectrophotometer). IR (KBr): 3642.22 cm⁻¹ (OH stretching), 1731.96 cm⁻¹ (ketone), 2939.3 cm⁻¹ (aromatic C-H stretching), 1641.31 cm⁻¹ (C=C in aromatic system), 2939.3 cm⁻¹ (C-H stretching aliphatic).

Discussion

The anti-inflammatory and analgesic activities of leaves of P. zeylanica were investigated in the present study. The carrageenan test was selected because of its sensitivity in detecting orally active anti-inflammatory agents. Non-steroidal anti-inflammatory agents (NSAIDs) such as the reference diclofenac used in this study are known to inhibit cyclooxygenase enzymes I and II, which are implicated in the production of inflammation-mediating agent prostaglandin E2 (PGE2) from arachidonic acid (Calvo, 2006). The first phase (0–2.5 h) after the injection of carrageenan results from the concomitant release of mediators such as histamine, serotonin, and kinins on the vascular permeability. The second phase is correlated with the elevated production of prostaglandins, oxygen-derived free radicals, and the production of inducible cyclooxygenase (Menasse et al., 1978). The pattern of anti-inflammatory activity exhibited by these extracts and fractions tends to suggest that the activity may wholly or in part be mediated by cyclooxygenase enzymes I and II inhibition. The reduction of the anti-inflammatory process by the petroleum ether extract, acetone extract, and fractions F₂ and F₃, obtained during 3–4 h, is probably related to a reduction in the synthesis and release of prostaglandins, rather than preformed inflammatory agents.

The hot plate test has been found to be suitable for the evaluation of centrally acting drugs, while the formalin test is another pain model, which assesses the way the animal responds to moderate continuous pain generated by injured tissue. The effect of F₃ in prolonging the pain sensation indicated the central activity of the fraction. The formalin test produces a distinct biphasic response, and different analgesics may act differently in the early and later phases of the test. Therefore, the test can clarify the possible mechanism of the antinociceptive effect of a proposed analgesic (Daud et al., 2006). Centrally acting drugs inhibit both phases, while peripherally acting drugs inhibit only the later phase. The effect of extracts (petroleum ether and acetone) and fraction (F₂) only in the later phase supports the anti-inflammatory action of the extract.

In our preliminary phytochemical investigation, we revealed the presence of fats, steroids, and naphthaquinones in the petroleum ether extract, while the acetone extract contained tannins, flavonoids, triterpenoids, and naphthaquinones. The anti-inflammatory and analgesic activities of the isolated plumbagin were further confirmed. The observed anti-inflammatory and analgesic activities of the extracts can be attributed to the presence of naphthaquinone, plumbagin as one of the active constituents, with tannins, flavonoids, and triterpenoids, playing a lead role in protection against inflammation and analgesia.

Conclusion

The present study indicates that the herb possesses anti-inflammatory and analgesic properties, which are mainly attributed to the presence of plumbagin, and these findings provide pharmacological support to the folkloric uses of the plant in the treatment of inflammation.

Acknowledgement

The authors are thankful to the Director, BRNCP, for providing all the necessary facilities to carry out this research work.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.