Journal of Ethnopharmacology 238 (2019) 111827

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm

Acute anti-inflammatory and anti-nociceptive activities of crude extracts, T

alkaloid fraction and evolitrine from Acronychia pedunculata leaves
W.M.K.M. Ratnayakea, T.S. Suresha, A.M. Abeysekerab, N. Salimc, U.G. Chandrikaa,∗
a
Department of Biochemistry, Faculty of Medical Sciences, University of Sri Jayewardenepura, Gangodawila, Nugegoda, Sri Lanka
b
Department of Chemistry, Faculty of Applied Sciences, University of Sri Jayewardenepura, Gangodawila, Nugegoda, Sri Lanka
c
Department of Botany, Faculty of Applied Sciences, University of Sri Jayewardenepura, Gangodawila, Nugegoda, Sri Lanka

ARTICLE INFO ABSTRACT

Keywords: Ethnopharmacological relevance: Acronychia pedunculata (family: Rutaceae) is one of the commonly used med-
Anti-inflammatory icinal plants in Sri Lankan traditional medicine. Different parts of this plant are used for the treatment of in-
Acronychia pedunculata flammatory conditions in the form of medicinal oils and herbal porridge.
Rutaceae Aim of the study: The present study aimed to evaluate the anti-nociceptive activity and anti-inflammatory ac-
Evolitrine
tivity with their mechanisms and the acute toxicity of crude extracts of the fresh leaves of A. pedunculata for
scientific validation of the ethnopharmacological claims for this plant. Further, attention has been focused on the
isolation of active compounds from active fractions of the crude extracts.
Materials and methods: The acute anti-inflammatory effect of the aqueous (AELA) and 70% ethanol crude ex-
tracts (EELA) and alkaloid fraction of A. pedunculata leaves were evaluated by the determination of inhibition of
hind paw oedema induced by carrageenan in Wistar rats. Evolitrine was identified as the major alkaloid with
significant bioactivities by column chromatography and NMR. The anti-nociceptive and anti-histamine activities
of EELA and evolitrine were evaluated by acetic acid induced writhing and wheal formation tests respectively. In
addition, in-vitro (2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay) and in-vivo (lipid peroxidation assay) anti-oxi-
dant activity, nitric oxide (NO) inhibitory activity and acute toxicity of EELA were evaluated.
Results: Acute anti-inflammatory activity of AELA and EELA were dose-dependent. EELA was more active than
AELA. The 200 mg/kg body weight (b. w.) dose of EELA was found as the minimum effective dose with max-
imum inhibition (78%) of oedema at 5th hour compared to the negative control (p < 0.05). Evolitrine was
isolated and identified as an active anti-inflammatory and analgesic compound from active alkaloid fraction of
EELA. Evolitrine showed activity enhancement when compared with crude EELA. The anti-inflammatory and
analgesic activities of evolitrine (50 mg/kg b. w.) were comparable to that of reference drugs indomethacin
(5 mg/kg b. w) and acetylsalicylic acid (100 mg/kg b. w.). The significant (p < 0.05) anti-histamine activity,
DPPH scavenging in-vitro anti-oxidant activity, in-vivo lipid peroxidation inhibitory activity in-vivo, NO inhibitory
activity of EELA as compared with relevant negative controls, were identified as probable mechanisms which
mediated its anti-inflammatory action. Further, EELA showed a high safety margin in the limited dose acute
toxicity study.
Conclusion: The findings of the current study rationalize the usage of leaves of A. pedunculata in Sri Lankan
traditional medicine as an analgesic and anti-inflammatory agent. Possible mechanisms mediating this activity
included anti-histamine, anti-oxidant and NO inhibitory activities. Evolitrine is the major analgesic and anti-
inflammatory compound isolated from the active alkaloid fraction of EELA.

1. Introduction resulting from the original insult (Kumar et al., 2007). It is character-
ized by heat, redness, pain, swelling and loss of function (Chandra et al.,
Inflammation is a protective response intended to eliminate the 2012). Enzyme activation, extravasation of fluid, release of mediators,
initial cause of cell injury as well as the necrotic cells and tissues cells migration, breakdown and repair of tissue are the biochemical

Corresponding author.
∗

E-mail addresses: kratnayake1984@gmail.com (W.M.K.M. Ratnayake), sugandhika@sjp.ac.lk (T.S. Suresh), ajitabey@gmail.com (A.M. Abeysekera),
nsalim7@yahoo.com (N. Salim), chandri@sjp.ac.lk (U.G. Chandrika).

https://doi.org/10.1016/j.jep.2019.111827
Received 7 January 2019; Received in revised form 19 March 2019; Accepted 19 March 2019
Available online 22 March 2019
0378-8741/ © 2019 Elsevier B.V. All rights reserved.
W.M.K.M. Ratnayake, et al. Journal of Ethnopharmacology 238 (2019) 111827

events which lead to inflammation. Even though inflammation is a body was also evaluated. In addition, the anti-histamine, anti-oxidant (in-
protective mechanism, inadequate control and inappropriate as in the vitro and in-vivo) and NO scavenging activities were also investigated to
case of against self-tissue, it can become the cause of injury and diseases explore the possible mechanisms of action by which A. Pedunculata
(Kumar et al., 2007). Steroids and non-steroidal anti-inflammatory exerts its anti-inflammatory effect. Although there have been no reports
drugs (NSAIDs) are the widely used allopathic drugs in the management on toxicity in the ethnomedical usage of this plant an acute toxicity
of inflammatory conditions. These drugs exert adverse effects such as study was carried out to evaluate its safety.
gastric irritation which lead to the formation of gastric ulcers. Fur-
thermore, these have shown limited success as treatments for all forms 2. Materials and methods
of inflammatory conditions (Arawwawala et al., 2010). Hence, there is
a need for continuous research focusing on the discovery of novel anti- 2.1. Ethical clearance
inflammatory compounds with lesser or no adverse effects. In this
context, the investigation of plant-based anti-inflammatory agents is an The protocol for animal experiments was approved by the Ethics
active area of current research. Review Committee of the Faculty of Medical Sciences, University of Sri
Acronychia pedunculata (Family- Rutaceae) is one of the commonly Jayewardenepura, Sri Lanka (No. 30/14, 35/15). International guide-
used medicinal plants in Sri Lankan traditional systems of medicine. It lines and recommendations of the Federation of European Laboratory
is generally known as “Ankenda” in Sinhala, claw-flowered laurel in Animal Science Associations (FELASA) were followed for the handling
English and “Kattukanni” in Tamil (Jayaweera, 1982). Leaves, roots, of animals. Assays were carried out at the Animal House and the
barks and fruits of this plant have been used in folk medicine of dif- Department of Biochemistry of University of Sri Jayewardenepura, Sri
ferent regions in the world for the treatment of diarrhoea, cough, Lanka.
asthma, ulcers, itchy skin, scales, pain, swellings, rheumatism and
disorders with involvement of the inflammatory processes (Jayaweera, 2.2. Animals
1982). Also, they are used as anti-pyretic and antihaemorrhagic agents
as well as aphrodisiacs (Han et al., 2004). Various topical applications Healthy adult Wistar albino rats weighing 150–200 g were pur-
as well as orally administered drugs prepared from leaves, barks and chased from Medical Research Institute, Colombo 8, Sri Lanka. Male
roots of this plant, are used in Sri Lankan traditional medicine. It can rats were used for all the experiments except the toxicity study. Rats
also be found in countries such as Indonesia, Malaysia, Southern China, were housed under standard conditions with a natural light-dark cycle
Hong Kong and Philippines (Jayaweera, 1982; Pathmasiri et al., 2005; and fed ad libitum with standard diet and clean fresh water. The animals
Su et al., 2003). were acclimatized for at least one week to the laboratory conditions
A search of the literature on this plant revealed only a few studies on before commencing experiments. Except at the period of experimental
the chemical constituents and biological activities of A. pedunculata. procedures the animals were handled only during cage cleaning. In all
Two furoquinoline alkaloids, kokusaginine (0.1% from the leaves) and experimental models, six animals were used in each group. All rats were
evolitrine (0.05% from the timber) have been reported from the Sri deprived of food overnight (9.00 p. m to 7.00 a. m prior to conducting
Lankan variety of this plant (De Silva et al., 1979). However, the the experiment.
phytochemical screening of leaves and stem of Indian variety has shown
the absence of alkaloids (Gireesha and Raju, 2016). Hence, these two 2.3. Chemicals
phytochemicals may be tentatively considered as chemotaxonomic
markers of A. pedunculata species of Sri Lanka. Carrageenan and histamine dihydrochloride, 2, 2-diphenyl-1-pi-
Wu and co-workers have shown that the methanol extract of the crylhydrazyl (DPPH), thiobarbituric acid and carboxymethyl cellulose
stem and root bark of A. pedunculata has significant cytotoxicity in the were purchased from Sigma-Aldrich Company (St. Louis MO, USA).
human KB tissue culture assay. They isolated the polyphenolic dimeric Indomethacin, chlorpheniramine and acetyl salicylic acid were pur-
acetophenone, acrovestone, as a cytotoxic principle from the extract by chased from the State Pharmaceutical Corporation, Sri Lanka. All other
bioassay-directed fractionation (Wu et al., 1989). Lesueur et al. (2008) chemicals and reagents were of analytical grade.
have shown that essential oil isolated from the aerial parts, possess
broad spectrum anti-microbial activity against various bacteria, parti- 2.4. Plant material
cularly Salmonella enterica and Staphylococcus epidemidis. Kozaki et al.
(2014) and co-workers worked on dried leaves and twigs of this plant Mature fresh A. pedunculata leaves were collected from a tree in the
and they have shown the polymerase α and β inhibitory action of new Colombo district (Latitude 6.8549, Longitude 79.9684), Sri Lanka. It
acetophenone, Acronyculatin F, isolated from it. Pathmasiri et al. was authenticated and a voucher specimen (KMR002) was deposited at
(2005) showed that acrovestenol which was isolated from a di- National Herbarium, Department of National Botanic Garden,
chloromethane extract of the bark of this plant inhibited cycloox- Peradeniya, Sri Lanka.
ygenase-2 (COX-2) with an IC50 value approximately thirteen times less
than the COX-2 inhibitory reference compound (NS 398). 2.5. Preparation of crude leaf extracts of A. pedunculata
Although a number of acetophenone dimers (acrovestone, acro-
vestenol, acropyrone, acropyranol A, acropyranol B, acrofolione A, The collected fresh leaves were washed and cut into pieces. The
acrofolione B), an acetophenone monomer (acronyline) and fur- aqueous extract of leaves of A. pedunculata (AELA) was prepared by
oquinoline alkaloids (dicatamine, pteleine, evolitrine, kokusaginine) refluxing 100 g of small pieces of fresh leaves with distilled water
have been isolated from timber, bark and leaves of A. pedunculata (De (500 mL) for 2 h. The extract was filtered and the brownish filtrate was
Silva et al., 1979; Wu et al., 1989; Pathmasiri et al., 2005; Kouloura freeze dried. The extract yielded 2.6 g of yellowish brown coloured
et al., 2012), there is a paucity of literature about the biological activity freeze dried powder (2.6%). The 70% ethanol extract of leaves of A.
of those compounds. To the best of our knowledge, no work has been pedunculata (EELA) was made by refluxing 100 g of small pieces of fresh
reported on in-vivo anti-inflammatory and anti-nociceptive activities of leaves with 70% ethanol (500 mL) for 2 h. The extract was filtered. The
the extracts of this plant. The present study was focused on in- dark greenish filtrate was evaporated under reduced pressure to dry-
vestigating the anti-inflammatory activity of the aqueous and 70% ness. The extract yielded 5.3 g of blackish brown semi-solid material
ethanol crude extract of leaves of the A. pedunculata and carrying out (5.3% w/w).
activity guided separation of active compounds of extract. Since oc- Both AELA and EELA were placed in airtight containers and stored
curring of pain is a response to inflammation, anti-nociceptive activity in the refrigerator at 4 °C until used in the bioassay. The AELA and EELA

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W.M.K.M. Ratnayake, et al. Journal of Ethnopharmacology 238 (2019) 111827

were suspended in 0.5% carboxymethyl cellulose (CMC) at a required experimental model for the evaluation of acute anti-inflammatory effect
concentration in l mL solution for oral administration to rats via oral as described by Winter et al. (1962) with some modifications. Wistar
feeding needles. rats were randomly divided into six groups and baseline values of the
left hind paw volume were taken at the zero hour using a plethysm-
2.6. Phytochemical screening ometer (Letica Scientific Instruments, Barcelona, Spain). The negative
and positive control groups were administered with 0.5% (w/v) CMC
The EELA was subjected to qualitative phytochemical screening for and indomethacin in 0.5% (w/v) CMC at 5 mg/kg b. w. respectively.
alkaloids, saponins, flavonoids, unsaturated sterols, triterpenes, leu- The treated groups received two doses of AELA and EELA in 0.5% (w/v)
coanthocyanins, tannins and polyphenols using standard procedures CMC, i.e. 100 and 500 mg/kg b. w. The volume administered was kept
described in Fong et al. (1974). The EELA was tested by Wagner's and constant at 1 mL. After 1 h, 0.1 mL of 1% carrageenan suspension in
Mayer's reagents for alkaloids. The froth test, Liberman – Burchard test, 0.9% saline solution was injected using a 27 G needle and 1 mL syringe
Salkowski test, cyanidin test, gelatin salt reagent test and ferric chloride into the subcutaneous tissue of the left hind paw of all rats under mild
test were carried out to evaluate the presence of saponins, unsaturated anesthesia. Thereafter, the volume of the injected paw of each of these
sterols, triterpenes, flavonoids, tannins and polyphenols respectively. rats was measured using the plethysmometer at hourly intervals up to
5 h. The degree of oedema was calculated by the paw volume increase
2.7. Alkaloid fraction of EELA (V0 – Vt), where V0 is the paw volume for each group before injection of
carrageenan (at 0 h) and Vt is the paw volume for each group at time “t”
The alkaloid fraction of EELA was extracted as follows. (at 1, 2, 3, 4 and 5 h). The percentage inhibition of oedema was cal-
The fresh leaves were dried in the oven at 40 °C to constant weight culated by the following equation.
and powdered in a mechanical grinder. The dried and milled leaves
[ONC OT ]
(100 g) were refluxed with 70% ethanol (500 mL) to obtain EELA as Percentage inhibition of oedema = × 100
ONC
described in section 2.5. The extract yielded 7.4 g of a blackish brown
semi-solid (7.4 w/w %). This was repeatedly extracted with hot HCl Where, ONC is the mean paw oedema of negative control at time “t” and
(2 N) until the extract no longer gave a positive result with Wagner OT is the mean paw oedema in treated groups at time “t”.
reagent and Mayer's reagent. The combined acid extract was washed
with chloroform (3 × 150 mL) using a separatory funnel. The acid ex- 2.9.2. Estimation of an optimum effective dose of EELA for acute anti-
tract was then basified with excess ammonia and extracted with inflammatory action
chloroform (3 × 150 mL). The combined chloroform extract was eva- The method given in section 2.9.1, was used to evaluate the effec-
porated to dryness under reduced pressure and the alkaloid fraction was tive dose of EELA in acute anti-inflammatory action. The treated groups
obtained as a brown coloured solid (0.14 g). The alkaloid fraction was were administered with EELA in 0.5% (w/v) CMC at the doses of 100,
analysis by thin layer chromatography (TLC) on silica with chloroform: 200, 300 and 500 mg/kg b. w.
ethyl acetate (9: 1 v/v) as the solvent system revealed the presence of a
major alkaloid and traces of four other minor alkaloids. The visuali- 2.9.3. Evaluation of acute anti-inflammatory activity of alkaloid fraction of
zation of alkaloids was achieved by spraying the TLC plate with the EELA
Dragendroff reagent. The acute anti-inflammatory activity of the alkaloid fraction of
EELA (AF-EELA) was also evaluated as described in section 2.9.1. The
2.8. Isolation and identification of evolitrine treated groups were administered EELA and AF-EELA in 0.5% (w/v)
CMC at the dose of 100 mg/kg b. w.
The major alkaloid present in the alkaloid fraction of A. pedunculata
leaves was isolated using a method described by De Silva et al. (1979) 2.9.4. Evaluation of acute anti-inflammatory activity of evolitrine
for the extraction of kokusaginine with some modifications. The brown The method given in section 2.9.1, was also used to evaluate acute
coloured alkaloid fraction was recrystallized with petroleum ether anti-inflammatory activity of evolitrine, at the doses of 25, 50, 75 and
(60–80 °C) to obtain white rod-shaped crystals. However, TLC analysis 100 mg/kg b. w. in 0.5% (w/v) CMC. The increase in paw volume and
revealed the presence of two alkaloids in the crystals. The isolation of percentage inhibition of oedema was calculated as described in section
the major alkaloid from the mixture (0.103 g) was done by column 2.9.1.
chromatography on basic alumina (200 g, 56 cm × 2.5 cm) using di-
chloromethane as the mobile phase (flow rate 5 mL/min). The fractions 2.10. Evaluation of the anti-nociceptive activity of EELA and evolitrine
(1.5 mL) were collected and TLC analysis was done to identify the
presence of alkaloids. The fractions containing the major alkaloid Anti-nociceptive activity was evaluated using the acetic acid-in-
(Fractions 38–60) were pooled and the solvent was evaporated. It duced writhing method as described by Somchit et al. (2004). Wistar
yielded the major alkaloid as a white coloured powder (86 mg, 0.086% rats were randomly divided into three groups (n = 6/group). The ne-
w/w from dried leaves). Rechromatography of fractions 61–76, yielded gative and positive control groups received 0.5% (w/v) CMC and
a further 10 mg of the major alkaloid (0.01% w/w from dried leaves). 100 mg/kg acetyl salicylic acid in 0.5% (w/v) CMC respectively. The
The purity of the isolated major alkaloid was checked by TLC on silica. experimental group was given EELA in 0.5% (w/v) CMC at the dose of
The melting point was also determined. The molecular mass of the 200 mg/kg b. w., the minimum effective dose with maximum inhibition
compound was determined using a MS 6130 attached to HPLC-1260 of oedema as determined in the previous assay. After 1 h of oral ad-
infinity, Quat Pump VL. The NMR spectra data were obtained from ministration of vehicle (negative control group), standard drug (posi-
NMR Burke Ascend ™ 400 Hz. The structure of the compound estab- tive control group) or plant extract (treated group), writhing was in-
lished as evolitrine by comparison of its 1H NMR and 13C NMR with the duced by the administration of 10 mL kg−1 b. w. of acetic acid solution
literature (Pusset et al., 1991). (0.6% v/v) in normal saline, intraperitoneally. After 2 min of acetic acid
injection, the rats were observed through a transparent box and the
2.9. Evaluation of acute anti-inflammatory activity on carrageenan-induced number of writhes was counted for a period of 20 consecutive minutes.
rat paw oedema The writhing movements were measured as a contraction of the ab-
dominal muscles accompanied by stretching of the hind limbs. Anti-
2.9.1. Evaluation of acute anti-inflammatory activity of AELA and EELA nociceptive activity was calculated compared to the respective controls
Carrageenan-induced rat hind paw oedema test was used as an by the following formula.

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W.M.K.M. Ratnayake, et al. Journal of Ethnopharmacology 238 (2019) 111827

Percentage anti nociceptive activity =

WN.C WT
× 100 samples vs. percentage radical scavenging activity.
WN.C
2.12.2. Evaluation of the in-vivo anti-oxidant activity of EELA by lipid
Where, W N.C is the mean number of writhes for the negative control
peroxidation assay
group and W T is the mean number of writhes for the group treated with
The in-vivo anti-oxidant activity was evaluated by lipid peroxidation
the plant extract or the standard drug.
assay as described by Kumari et al. (2014) with some modifications.
In order to evaluate the anti-nociceptive activity of evolitrine, a
Wistar rats were randomly divided into two groups (n = 6/groups). The
dose of 50 mg/kg b. w. evolitrine in 0.5% (w/v) CMC was administered
negative control and test groups were orally administered 0.5% (w/v)
for the treated group and the experiment was repeated.
CMC and 200 mg/kg b. w. dose of EELA in 0.5% (w/v) CMC for 28
consecutive days as a single daily dose. The volume administered was
2.11. Evaluation of the anti-histamine activity of EELA and evolitrine kept constant at 1 mL. On 29th day blood was drawn from rats’ lateral
tail vein following 12 h fast and serum was separated to conduct the
Anti-histamine activity was evaluated as described by Kumari et al. lipid peroxidation assay. Aliquots of the 200 μL of serum were depro-
(2014) employing the wheal formation test. Wistar rats were randomly teinized by adding 1 mL of 14% trichloroacetic acid and 1 mL of 0.6%
divided into three groups (n = 6/group). The fur on the left lateral side thiobarbituric acid (TBA). The mixture was heated in a water bath for
of the back of each rat was shaven. After 24 h, the negative and positive 30 min to complete the reaction and then cooled on ice for 5 min. The
control groups received 0.5% (w/v) CMC and chlorpheniramine in absorbance of the coloured product was measured at 535 nm with a uv
0.5% (w/v) CMC at the dose of 0.67 mg/kg b. w. respectively. The spectrophotometer. The concentration of malondialdehyde was calcu-
treated group received EELA (200 mg/kg b. w.) in 0.5% (w/v) CMC lated by using the formula of C = A/∑L; where, A is the absorbance, ∑ is
which was found as the effective dose in acute anti-inflammatory as- the molar extinction coefficient of the complex formed by mal-
says. After 1 h of oral treatment, histamine dihydrochloride in saline ondialdehyde and TBA (1.56 × 105 M−1 cm−1) (Devasagayam et al.,
(50 μL of 200 μg/mL solution) was injected subcutaneously into the skin 2003), C is the concentration of malondialdehyde, L is the path length.
where the fur was shaven. Two minutes after the subcutaneous injec-
tion, the area of the wheal formed was measured by drawing each 2.13. Evaluation of nitric oxide radical scavenging activity of EELA
wheal by placing a transparent sheet on the area of the shaven skin.
Anti-histamine activity was expressed as percentage inhibition of wheal In this assay, nitric oxide generated from the sodium nitroprusside
formation calculated as follows. (SNP), was measured by the Greiss reaction as described by Jagetia
ANC AT et al. (2004). An aliquot of 2 mL of SNP (10 mM) was mixed with 500 μL
Percentage inhibition of wheal formation = × 100
ANC of phosphate buffered saline (50 mM) and 500 μL of EELA (50, 100,
200, 250, 300 and 500 μg/mL) or quercetin (20, 40, 80, 100 and
Where, ANC is the mean area of the wheal for the negative control group 200 μg/mL). The same reaction mixture, without a test sample but, with
and AT is the mean area of the wheal for the group treated with the an equivalent amount of ethanol served as a negative control. To avoid
EELA or standard drug. the interferences of absorbance by the colour of the test sample (EELA
This method was repeated to evaluate anti-histamine activity of or quercetin), a blank sample was prepared by mixing 500 μL of the test
evolitrine (50 mg/kg b. w.) in 0.5% (w/v) in CMC by oral administra- sample with 2.5 mL of PBS. All samples were incubated at 25 °C for
tion for the treated group. 150 min. After the incubation period, an aliquot of 1 mL was pipetted
and mixed with 1 mL of freshly prepared Greiss reagent [A 1% sul-
2.12. Evaluation of anti-oxidant activity of EELA phanilamide in 5% phosphoric acid (1.01 g sulphanilamide (assay 99%)
and 5.68 mL of phosphoric acid (assay 88%) in 100 mL of distilled
2.12.1. In-vitro antioxidant activity of EELA by DPPH radical scavenging water) and 0.1% N-(1-naphthyl)-ethylene diamine dihydrochloride
assay (0.1 g in 100 mL of distilled water) were separately prepared and mixed
The in-vitro anti-oxidant activity was assessed based on the together (1: 1 v/v) before the analysis]. The absorbance of the chro-
scavenging activity of the stable DPPH free radical as described by mophore formed, was measured at 546 nm following an incubation
Loizzo et al. (2010) . An aliquot of 1.5 mL of 0.25 mM DPPH solution period of 30 min. All the tests were performed in triplicate for each
was mixed with 1.5 mL of EELA (3.125, 6.25, 12.5, 25 and 50 μg/mL). concentration. Anti-oxidant activity was measured in terms of nitric
Similarly, a series of gallic acid (0.5, 1, 2, 3, 4 and 5 μg/mL) used as a oxide scavenging activity and the percentage scavenging activity was
reference substance. To avoid the interferences of absorbance by the calculated using the following formula.
colour of the test sample, the blank sample was prepared by mixing
[A 0 AT]
1.5 mL of methanol and 1.5 mL of EELA for each concentration. The Scavenging activity (%) = × 100
A0
reaction mixture was allowed to reach the steady state at room tem-
perature in the dark for 30 min after which the absorbance at 517 nm Where, A0 is the absorbance of the negative control and AT is the ab-
was measured. Methanol was used as the negative control. All the tests sorbance of the test sample.
were performed in triplicate for each concentration. Anti-oxidant ac- The whole experiment was repeated thrice separately and the NO
tivity was measured in terms of radical sacvenging activity and the scavenging activity of test samples were expressed as a mean of EC50
percentage scavenging effect was calculated using the following for- (μg/mL), which is defined as the mean concentration of the anti-oxidant
mula. required to lower the radical concentration by 50% in each experiment.
[A 0 AT] It was determined by using the graph plotted with the mean con-
Scavenging activity (%) = × 100 centration of triplicates of each test samples vs. percentage NO
A0
scavenging activity.
Where, A0 is the absorbance of the negative control and AT is the ab-
sorbance of the test sample (EELA or gallic acid). The entire experiment 2.14. Limited dose acute toxicity study of EELA
was repeated thrice. The radical scavenging activity of test samples,
was expressed as a mean of EC50 (μg/mL), which is defined as the mean The limit dose test was performed at the dose level of 5000 mg/kg b.
concentration of the anti-oxidant required to lower the initial DPPH w., in compliance with the Organization for Economic Co-operation and
concentration by 50% in each experiment. It was determined by using Development (OECD) guidelines No. 425, acute oral toxicity – fixed
the graph plotted with the mean concentration of triplicates of each test dose method (OECD, 2008). Female Wistar rats were randomly divided

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W.M.K.M. Ratnayake, et al. Journal of Ethnopharmacology 238 (2019) 111827

into two groups and fasted overnight with access only to water. Fol- Table 2
13
lowing the fast, control group and test group were orally administered C NMR chemical shifts for evolitrine.
0.5% (w/v) CMC and 5000 mg/kg b. w. of EELA in 0.5% (w/v) CMC Carbon Observed Reporteda
respectively.
All the animals were observed individually for mortality and be- 1 142.6 142.5
2 161.3 161.3
havioural changes such as salivation, diarrhoea, loss of hair, postural
3 102.0 102.2
abnormalities etc. for first 30 min, 1 h, 2 h, 4 h and 6 h after dosing and 4 157.5 157.3
thereafter once a day for 14 consecutive days. The mortality and clin- 5 116.9 116.8
ical signs, body weights and the food consumptions of rats were re- 6 123.6 123.6
corded during the period. All rats were sacrificed on the day 15 and 7 147.1 147.7
8 105.5 106.0
blood samples were also collected. The separated serum was used to
9 113.3 113.6
analyse biochemical parameters; alanine amino transferase (ALT), al- 10 104.9 104.9
kaline phosphatase (ALP), aspartate aminotransferase (AST), γ-glu- 11 142.5 142.5
tamyl transferase (γ-GT), urea and creatinine. OMe-4 59.0 59.0
OMe-7 55.4 55.5

2.15. Statistical analysis CDCl3, TMS as internal standard,δ, ppm.

a
Pusset et al. (1991).
All the results were subjected to descriptive statistics and expressed
as mean ± standard error of the mean (SEM). Data were analyzed by
one-way analysis of variance test (ANOVA) followed by Bonferroni test
method for post hoc analysis, using SPSS statistic 21 to determine the
significance of the difference between the experiment groups. p-va-
lues < 0.05 was considered as statistically significant.

3. Results

3.1. Phytochemical screening Fig. 1. The structure of the evolitrine.

The qualitative phytochemical screening of EELA showed strong

positive observations for alkaloids with Mayer's test and Wagner's test.
In addition, it revealed the presence of flavonoids, unsaturated sterols,
triterpenes, tannins and polyphenols.

3.2. Isolation and identification of evolitrine

Comparison the 1H NMR and 13C NMR spectra (Tables 1 and 2) with
published data (Pusset et al., 1991) clearly established the major al-
kaloid isolated as described in section 2.8 as evolitrine (Fig. 1). The LC- Fig. 2. The structure of the kokusaginine.
MS of the compound gave a clear peak for the [M+H]+ion at m/z 230.
Our result is at variance with the report by De Silva et al. (1979) that
kokusaginine (Fig. 2) is the major alkaloid in the petroleum extract of
peak at 2 h and then began to decline. All doses of AELA and EELA
the leaves of A. peduculata. However, evolitrine is reported by De Silva
showed the maximum percentage inhibition at the 5th h as did the
as the major alkaloid found in the petroleum extract of the timber of A.
standard drug indomethacin (Table 3). Both AELA and EELA at the dose
peduculata. We note that the melting point of evolitrine recorded by us
of 500 mg/kg b. w., showed significant (p < 0.05) reductions in paw
(129 °C) is higher than that reported by Pusset et al. (1991) (114 °C).
oedema as compared to the negative control. However, of the two ex-
tracts, only EELA showed a significant (p < 0.05) reduction at the
3.3. Effect of acute anti-inflammatory activity of AELA, EELA, AF-EELA lower dose of 100 mg/kg b. w. Hence, EELA was selected for further
and evolitrine on carrageenan-induced rat paw oedema studies and dose response study was carried out to find out the
minimum effective dose as shown in Table 4.
The subcutaneous injection of carrageenan into the hind footpad of All the test doses of EELA had significant acute anti-inflammatory
rats produced local oedema in the following 1 h that progressed to its activity as shown by significantly (p < 0.05) reduced paw oedema
compared to the negative control. The maximum percentage inhibition
Table 1 on oedema was observed at the 5th hour as did indomethacin. As shown
1
H NMR chemical shifts for evolitrine.
in Table 2, there is a significant (p < 0.05) increase in the inhibition of
Protons Observed Reporteda oedema, when the dose is increased from 100 to 200 mg/kg b. w. EELA.
However, the differences in inhibition of oedema among the 200 mg/kg
5 8.15 d (9.28) 8.15 d (9.3)
6 7.1 dd (9.28, 2.5) 7.09 dd (9.3, 2.6)
b. w. dose and the higher doses were not statistically significant
8 7.35 d (2.5) 7.33 d (2.6) (p > 0.05). Hence, the dose of 200 mg/kg b. w. of EELA was identified
10 7.06 d (2.8) 7.05 d (2.8) as the minimum dose which gives maximum inhibition of paw oedema
11 7.58 d (2.8) 7.57 d (2.8) and it was used to evaluate the activity of A. pedunculata leaf extract on
OMe-4 4.44 s 4.43 s
different anti-inflammatory mechanisms.
OMe-7 3.96 s 3.95 s
The carrageenan-induced paw oedema model was also used to
CDCl3, TMS as internal standard,δ, ppm, coupling constant in Hz are given in evaluate the acute anti-inflammatory effect of AF-EELA. The results
parenthesis. showed that the dose of 100 mg/kg b. w. of EELA treated group as well
a
Pusset et al. (1991). as AF-EELA treated group have significant (p < 0.05) acute anti-

5
W.M.K.M. Ratnayake, et al. Journal of Ethnopharmacology 238 (2019) 111827

Table 3
Acute anti-inflammatory effect of AELA and EELA on the carrageenan-induced rat hind paw oedema in Wistar rats.
Group Increase in paw volume at different time intervals (mL) ± SEM, (Percentage Inhibition)d

1 Hour 2 Hour 3 Hour 4 Hour 5 Hour

3 3 3 3
Group 1 0.30 ± 0.03 0.61 ± 0.03 0.54 ± 0.02 0.48 ± 0.02 0.38 ± 0.02
b b b b
Group 2 0.20 ± 0.02 (33%) 0.32 ± 0.02 (48%) 0.20 ± 0.02 (63%) 0.10 ± 0.02 (79%) 0.04 ± 0.01 (90%)
2 3 3 3
Group 3 0.24 ± 0.04 (20%) 0.50 ± 0.05 (18%) 0.42 ± 0.05 (22%) 0.36 ± 0.06 (25%) 0.28 ± 0.05 (26%)
a b 1 a 1 b 1
Group 4 0.22 ± 0.02 (26%) 0.43 ± 0.02 (30%) 0.35 ± 0.02 (35%) 0.28 ± 0.02 (42%) 0.20 ± 0.02 (47%)
b b b b
Group 5 0.21 ± 0.01 (30%) 0.35 ± 0.02 (43%) 0.25 ± 0.02 (54%) 0.16 ± 0.02 (67%) 0.11 ± 0.02 (71%)
a b b b b
Group 6 0.18 ± 0.01 (40%) 0.32 ± 0.01 (47%) 0.22 ± 0.02 (59%) 0.12 ± 0.01 (75%) 0.04 ± 0.01 (90%)

a
p < 0.05; b p < 0.001 compared with negative control, 1 p < 0.05; 2 p < 0.01; 3 p < 0.001 compared with positive control.
Group 1: Negative control group (0.5% CMC), Group 2: positive control group (5 mg/kg b. w., indomethacin in 0.5% CMC), Group 3: Treated group (100 mg/kg b. w.,
AELA in 0.5% CMC), Group 4: Treated group (100 mg/kg b. w., EELA in 0.5% CMC), Group 5: Treated group (500 mg/kg b. w., AELA in 0.5% CMC), Group 6: Treated
group (500 mg/kg b. w., EELA in 0.5% CMC).
d
Difference between measured paw volume and paw volume before carrageenan injection Figures in parenthesis indicate percentage inhibition of oedema.

inflammatory activity as compared to the negative control group 3.4. Effect anti-nociceptive activity of EELA and evolitrine
(Table 5). Similar to the indomethacin (5 mg/kg b. w.), both groups
showed the maximum inhibition of oedema at the 5th h after the in- As shown in Table 7 and Table 8, the EELA and evolitrine restrained
jection of carrageenan. Although the treated doses of EELA and AF- the writhing reflux induced by acetic acid with an inhibition percentage
EELA are higher than that of indomethacin, the maximum inhibition of 25% and 63% respectively. This inhibition was compared with the
oedema was less than that of indomethacin. Hence, acute anti-in- positive control group which was received acetyl salicylic acid. There
flammatory action of both EELA and AF-EELA was lower than that of was a significant (p < 0.05) difference between rat groups treated with
indomethacin. However, the maximum inhibition of oedema by AF- EELA and acetyl salicylic acid indicating that the anti-nociceptive ac-
EELA, was approximately twice as that of EELA. Hence, AF-EELA was tivity of EELA was less than acetyl salicylic acid.
identified as the active fraction in EELA and it was subjected to ex- Evolitrine inhibits writhes by 63% compared with 25% by EELA at
periments for isolation of compounds. four times the dose of evolitrine. Thus, there is a considerable en-
Evolitrine was identified as the major alkaloid present in the active hancement of activity in evolitrine when compared with EELA. Hence,
alkaloid fraction of EELA and the optimally effective anti-inflammatory it was identified as the major analgesic compound present in the A.
dose of evolitrine was determined by carrageenan paw oedema model. pedunculata leaves. In contrast to the results of EELA, there was no
The results revealed that all the test doses of evolitrine significantly significant (p > 0.05) difference between the positive control and
(p < 0.05) reduced paw oedema as compared to the negative control evolitrine treated group. Further, acetyl salicylic acid inhibits writhes
(Table 6). Hence, all the test doses of evolitrine have significantly by 55% by a dose at two times the dose of evolitrine. Thus, evolitrine
higher acute anti-inflammatory activity than the negative control. has a higher anti-nociceptive activity than the acetyl salicylic acid.
Further, all evolitrine doses showed their maximum percentage in-
hibition of oedema at the 5th h as did the indomethacin. Hence, the 3.5. Effect anti-histamine activity of EELA and evolitrine
results from this study strongly indicate the NSAIDs’ like activity of
evolitrine. The EELA and evolitrine significantly (p < 0.001) inhibited wheal
In addition, there is a significant difference (p < 0.05) in oedema formation on the skin of the rat after the injection of histamine with
between the highest (100 mg/kg b. w.) and lowest (25 mg/kg b. w.) 29% and 35% inhibition respectively as compared with the negative
doses of evolitrine. However, the difference between the doses of control group which received 0.5% (w/v) CMC. However, both of EELA
50 mg/kg b. w. and 100 mg/kg b. w. was not significant (p > 0.05). and evolitrine showed less anti-histamine activity as compared to the
Hence, the dose of 50 mg/kg b. w. of evolitrine was found as the standard drug, chlorpheniramine (Tables 9 and 10).
minimum effective dose with maximum inhibition of paw oedema. It is
interesting to note that percentage inhibition of oedema by effective 3.6. The DPPH radical scavenging assay
dose of evolitrine throughout the period of study, was comparable to
indomethacin which was the positive control. The EELA exhibited dose dependent DPPH scavenging activity with

Table 4
Acute anti-inflammatory effect of different doses of EELA on carrageenan-induced rat hind paw oedema in Wistar rats.
Group Increase in paw volume at different time intervals (mL) ± SEM, (Percentage Inhibition)d

1 Hour 2 Hour 3 Hour 4 Hour 5 Hour

1 3 3 3 3
Group 1 0.24 ± 0.03 0.37 ± 0.03 0.34 ± 0.05 0.25 ± 0.04 0.18 ± 0.04
c c c c
Group 2 0.17 ± 0.02 (30%) 0.18 ± 0.02 (51%) 0.10 ± 0.01 (71%) 0.06 ± 0.01 (76%) 0.02 ± 0.01 (89%)
b b b 2 b 1
Group 3 0.19 ± 0.04 (21%) 0.29 ± 0.05 (22%) 0.23 ± 0.02 (32%) 0.17 ± 0.08 (32%) 0.10 ± 0.03 (44%)
a c c c c
Group 4 0.16 ± 0.02 (33%) 0.22 ± 0.02 (41%) 0.12 ± 0.02 (65%) 0.06 ± 0.01 (76%) 0.04 ± 0.01 (78%)
a c c c c
Group 5 0.16 ± 0.02 (33%) 0.21 ± 0.03 (43%) 0.12 ± 0.01 (65%) 0.05 ± 0.01 (80%) 0.03 ± 0.02 (83%)
a c c c c
Group 6 0.15 ± 0.01 (38%) 0.20 ± 0.02 (46%) 0.11 ± 0.02 (68%) 0.05 ± 0.01 (80%) 0.03 ± 0.01 (83%)

a
p < 0.05; b p < 0.01; c p < 0.001 compared with negative control, 1 p < 0.05; 2 p < 0.01; 3 p < 0.001 compared with EELA treated group with 200 mg/kg b.
w dose.
Group 1: Negative control group (0.5% CMC), Group 2: positive control group (5 mg/kg b. w., indomethacin in 0.5% CMC), Group 3: Treated group (100 mg/kg b. w.,
EELA in 0.5% CMC), Group 4: Treated group (200 mg/kg b. w., EELA in 0.5% CMC), Group 5: Treated group (300 mg/kg b. w., EELA in 0.5% CMC), Group 6: Treated
group (500 mg/kg b. w., EELA in 0.5% CMC).
d
Difference between measured paw volume and paw volume before carrageenan injection Figures in parenthesis indicate percentage inhibition of oedema.

6
W.M.K.M. Ratnayake, et al. Journal of Ethnopharmacology 238 (2019) 111827

Table 5
Acute anti-inflammatory effect of AF-EELA on carrageenan-induced rat hind paw oedema in Wistar rats.
Group Increase in paw volume at different time intervals (mL) ± SEM, (Percentage Inhibition)d

1 Hour 2 Hour 3 Hour 4 Hour 5 Hour

1 1 1 1
Group 1 0.29 ± 0.04 0.56 ± 0.04 0.49 ± 0.05 0.31 ± 0.04 0.22 ± 0.04
1 c c 1 b 1 b 1
Group 2 0.18 ± 0.03 (38%) 0.26 ± 0.03 (54%) 0.14 ± 0.02 (71%) 0.09 ± 0.02 (72%) 0.02 ± 0.04 (91%)
a a a a
Group 3 0.22 ± 0.03 (24%) 0.42 ± 0.03 (25%) 0.30 ± 0.05 (39%) 0.20 ± 0.05 (36%) 0.12 ± 0.04 (45%)
c 1 c 1 a 1 b 1
Group 4 0.24 ± 0.03 (17%) 0.28 ± 0.04 (50%) 0.20 ± 0.02 (60%) 0.12 ± 0.03 (61%) 0.04 ± 0.01 (82%)

a
p < 0.05; b p < 0.01; c p < 0.001 compared with negative control, 1 p < 0.05 compared with EELA treated group with 100 mg/kg b. w dose.
Group 1: Negative control group (0.5% (w/v) CMC), Group 2: Positive control group (5 mg/kg b. w., indomethacin in 0.5% (w/v) CMC), Group 3: Treated group
(100 mg/kg b. w., EELA in 0.5% (w/v) CMC), Group 4: Treated group (100 mg/kg b. w., AF-EELA in 0.5% (w/v) CMC).
d
Difference between measured paw volume and paw volume before carrageenan injection Figures in parenthesis indicate percentage inhibition of oedema.

an EC50 value of 33.4 ± 0.1 μg/mL. This activity is less than standard Table 7
anti-oxidant, gallic acid, which has a EC50 of 2.4 ± 0.1 μg/mL. Anti-nociceptive effect of EELA on acetic acid induced writhing.
Group Number of writhes ± SEM (cm2) % Inhibition
3.7. In-vivo antioxidant activity by lipid peroxidation assay
# #
Group 1 61 ± 4 –
Group 2 **29 ± 2 52
The results have shown that serum MDA value of rats fed with EELA Group 3 #
*46 ± 3 25
(1.1 ± 0.3 μmol/L) was significantly (p < 0.05) lower than that of the
negative control group (1.8 ± 0.2 μmol/L). Values for area are expressed as mean ± SEM (n = 6/group) *P < 0.01,
**P < 0.001 compared with negative control; #p < 0.05, # #p < 0.001
compared with the positive control.
3.8. Nitric oxide radical scavenging assay
Group 1: Negative control group (0.5% (w/v) CMC), Group 2: positive control
group (100 mg/kg b. w., acetyl salicylic acid in 0.5% (w/v) CMC), Group 3:
The EELA exhibited dose-dependent nitric oxide scavenging activity
Treated group (200 mg/kg b. w., EELA in 0.5% (w/v) CMC).
with EC50 value of 209.7 ± 1.2 μg/mL and it was 108.6 ± 0.6 μg/mL
for quercetin.
Table 8
Anti-nociceptive effect of evolitrine on acetic acid induced writhing.
3.9. Limited dose acute toxicity study EELA
Group Number of writhes ± SEM % Inhibition

In the limited dose acute toxicity test, none of the rats in control and Group 1 #
67 ± 4 –
treated groups showed mortality and behavioural changes during the Group 2 *30 ± 2 55
period of observation (14 days). Further, both rat groups showed body Group 3 * 25 ± 2 63

weight gains and average food consumption throughout the study

Values for area are expressed as mean ± SEM (n = 6/group) *p < 0.001
period. Table 11 shows the clinical chemistry profiles of each rat groups compared with negative control; #p < 0.001 compared with the positive
and there was no significant difference (p > 0.05) between EELA control.
treated groups and healthy control group. Group 1: Negative control group (0.5% CMC), Group 2: positive control group
(100 mg/kg b. w., acetyl salicylic acid in 0.5% CMC), Group 3: Treated group
4. Discussion (50 mg/kg b. w., evolitrine in 0.5% CMC).

In the present study, an attempt has been made to evaluate the anti- weed (Necas and Bartosikova, 2013), which is a widely used phlogistic
inflammatory effect of 70% ethanol extract (EELA) and the aqueous agent which shows signs and symptoms of inflammation (Biradar et al.,
extract (AELA) of A. pedunculata using the carrageenan-induced rat 2010). Development of oedema in the paw of the rat following injection
hind paw oedema model. Further, this model was also used to identify of carrageenan is a multimediated phenomenon that liberates diverse
the active fractions and active compounds present in the EELA. mediators. It is believed to be a biphasic event (Vinegar et al., 1969).
Carrageenan is the sulphated polysaccharide obtained from a sea- The initial phase observed during the first 2 h is attributed to the release

Table 6
Acute anti-inflammatory effect of the evolitrine on the carrageenan-induced rat hind paw oedema in Wistar rats.
Group Increase in paw volume at different time intervals (mL) ± SEM, (Percentage Inhibition)d

1 Hour 2 Hour 3 Hour 4 Hour 5 Hour

1 3 3 3 3
Group 1 0.25 ± 0.02 0.50 ± 0.02 0.48 ± 0.02 0.41 ± 0.02 0.35 ± 0.01
a c c c c
Group 2 0.17 ± 0.02 (32%) 0.25 ± 0.03 (50%) 0.16 ± 0.02 (67%) 0.10 ± 0.02 (76%) 0.05 ± 0.02 (86%)
c 2 c 2 c 2 c
Group 3 0.20 ± 0.01 (20%) 0.32 ± 0.02 (36%) 0.22 ± 0.02 (54%) 0.15 ± 0.02 (63%) 0.09 ± 0.02 (74%)
c c c c c
Group 4 0.16 ± 0.01 (36%) 0.24 ± 0.01 (52%) 0.18 ± 0.01 (62%) 0.10 ± 0.01 (76%) 0.04 ± 0.01 (89%)
c c c c c
Group 5 0.14 ± 0.01 (44%) 0.21 ± 0.01 (58%) 0.14 ± 0.01 (71%) 0.07 ± 0.01 (83%) 0.03 ± 0.01 (91%)
b c c c c
Group 6 0.15 ± 0.01 (40%) 0.20 ± 0.01 (60%) 0.11 ± 0.01 (77%) 0.05 ± 0.01 (88%) 0.02 ± 0.01 (94%)

a
p < 0.05; b p < 0.01; c p < 0.001 compared with negative control, 1 p < 0.05; 2 p < 0.01; 3 p < 0.001 compared with evolitrine treated group with 100 mg/kg
b. w dose.
Group 1: Negative control group (0.5% CMC), Group 2: Positive control group (5 mg/kg b. w., indomethacin in 0.5% CMC), Group 3: Treated group (25 mg/kg b. w.,
evolitrine in 0.5% CMC), Group 4: Treated group (50 mg/kg b. w., evolitrine in 0.5% CMC), Group 5: Treated group (75 mg/kg b. w., evolitrine in 0.5% CMC), Group
6: Treated group (100 mg/kg b. w., evolitrine in 0.5% CMC).
d
Difference between measured paw volume and paw volume before carrageenan injection. Figures in parenthesis indicate percentage inhibition of oedema.

7
W.M.K.M. Ratnayake, et al. Journal of Ethnopharmacology 238 (2019) 111827

Table 9 evidence for the inhibitory activity of prostaglandin by A. pedunculata

Anti-histamine effect of EELA on wheal formation. leaves.
Group Area of wheal ± SEM (cm2) % Inhibition The current results showed that EELA is more active than the AELA
in the acute anti-inflammatory action. This activity increment could be
due to the possibility of median polar active compounds in the EELA is
b
Group 1 2.1 ± 0.2 –
Group 2 **0.7 ± 0.1 67
a
more soluble in ethanol than water. As EELA showed more activity, the
Group 3 * 1.5 ± 0.2 29
carrageenan-induced rat paw oedema model was used to find the op-
Values for area are expressed as mean ± SEM (n = 6/group) *p < 0.05, timal anti-inflammatory dose of EELA. The dose of EELA (200 mg/kg b.
**p < 0.001 compared with negative control; a p < 0.01, b p < 0.001 w.) was found as the minimum effective dose with maximum inhibition
compared with the positive control. of paw oedema (78%). Although no literature is available on in-vivo
Group 1: Negative control group (0.5% (w/v) CMC), Group 2: positive control anti-inflammatory activity of A. pedunculata leaves several other
group (0.67 mg/kg b. w., chlorpheniramine in 0.5% (w/v) CMC), Group 3: Rutaceae members have demonstrated similar activity i. e. 200 mg/kg
Treated group (200 mg/kg b. w., EELA in 0.5% (w/v) CMC). b. w of ethanol extract of Pleiospermium alatum (53%; Suky et al., 2011),
400 mg/kg b. w. of ethanol extract Aegle marmelosa (30%; Raju et al.,
Table 10 2016), 200 mg/kg b. w. of methanol extract Chloroxylon sweitenia (52%;
Anti-histamine effect of evolitrine on wheal formation. Sivakumar et al., 2005), 250 mg/kg b. w. of ethanol extract Murraya
Group Area of wheal ± SEM (cm2) % Inhibition koenigii (52%; Darvekar et al., 2011), 20 mg/kg b. w. of methanol ex-
tract Ruta graveolens (91%; Ratheesh and Helen, 2007) etc. According to
b
Group 1 2.0 ± 1.3 –
these literature reports, a comparatively stronger anti-inflammatory
Group 2 ** 0.9 ± 0.1 55
Group 3 a
* 1.3 ± 0.1 35 activity is evident in leaves of A. pedunculata (except the Ruta grave-
olens) among the Rutaceae members. Thus, these data provide scientific
Values for area are expressed as mean ± SEM (n = 6/group) *p < 0.01, evidence for the comparatively strong anti-inflammatory activity of
**p < 0.001 compared with negative control; a p < 0.05, b p < 0.001 leaves of A. pedunculata commonly used in Sri Lankan traditional
compared with the positive control Group 1: Negative control group (0.5% (w/ medicinal system.
v) CMC), Group 2: positive control group (0.67 mg/kg b. w., chlorpheniramine Many beneficial medicinal effects of plant materials typically result
in 0.5% (w/v) CMC), Group 3: Treated group (50 mg/kg b. w., evolitrine 0.5% from the combinations of phytochemicals which are derived as sec-
(w/v) CMC).
ondary products of the plants. Different plants are rich in different
secondary products such as alkaloids, flavonoids, sterols, terpenoids,
Table 11
phenolic acids and many others. The abundance of scientific evidence
Clinical chemistry data of Wistar rats in the limited dose acute oral toxicity
indicates that such compounds have biological effects such as anti-mi-
study.
crobial activity, antioxidant activity, anti-inflammatory activity, mod-
Parameters Group 1 Group 2 ulation of detoxification of enzymes, modulation of hormones, stimu-
lation of immune system etc. (Nyamai et al., 2016). Hence, in the
ALP (IU/L) 117.0 ± 2.7 121.4 ± 1.2
ALT (IU/L) 45.8 ± 2.3 48.6 ± 2.2 process of active compound isolation, phytochemical screening pro-
AST ((IU/L) 74.7 ± 1.8 74.7 ± 2.9 vides important information to conduct the study forward. The strong
Creatinine (mg/dL) 0.7 ± 0.1 0.6 ± 0.1 positive observations of EELA with Mayer's test and Wagner's test in-
γ- GT (IU/L) 22.6 ± 1.1 21.5 ± 1.3
dicated that it is rich with alkaloids. As, various types of alkaloids
Urea (mg/dL) 3.5 ± 0.2 3.9 ± 0.2
isolated from medicinal plants of family Rutaceae have shown anti-in-
Values for clinical chemistry data are expressed as mean ± SEM (n = 6/ flammatory activity (Iman et al., 2017; Kuo et al., 2004; Ratheesh et al.,
group). ∗p < 0.05 compared with healthy control. 2013) and as our test extract also is rich in alkaloids, the study was
Group 1: Healthy control group (Distilled water), Group 2: Treated group focused on evaluating the anti-inflammatory activity of the alkaloid
(5000 mg/kg b. w., EELA in 0.5% (w/v) CMC). fraction of EELA. As the alkaloid fraction showed the anti-inflammatory
activity enhancement at the 5th hour on carrageenan-induced oedema it
of histamine and serotonin (Vasudevan et al., 2006). The late phase of was identified as a fraction containing the active compounds. Subse-
oedema is linked to the eicosanoid release, neutrophil infiltration and quently, evolitrine was identified as the major alkaloid compound
release of neutrophil-derived mediators and production of free radicals present in EELA. The minimum effective dose with maximum inhibition
(Cuzzocrea et al., 1998). The chemical substances produced in both of oedema of evolitrine (50 mg/kg b. w.) is comparable with the acute
phases, cause an increase in vascular permeability and thereby promote anti-inflammatory activity of doses of 100 mg/kg b. w. of the crude
accumulation of fluid in tissues that accounts for oedema (Umukoro EELA and its alkaloid fraction, as well as with the standard drug in-
and Ashorobi, 2006). The results of the present study revealed that two domethacin (5 mg/kg b. w.) at the 5th hour of the experiment. Although
crude extracts (AELA and EELA) and the alkaloid fraction of A. ped- the dose of evolitrine (50 mg/kg b. w.) is lower than that of EELA
unculata leaves as well as evolitrine isolated from AF-EELA possess anti- (100 mg/kg b. w.) and the AF-EELA (100 mg/kg b. w.), it shows more
oedema activity in the early and in late phases of carrageenan induced inhibition of oedema. Hence, evolitrine from A. pedunculata leaves was
paw oedema model. These activities clearly indicate that all test ex- identified as an active compound with anti-inflammatory properties.
tracts and evolitrine may have inhibitory effects on chemical mediators Evolitrine has previously been isolated from the bark and leaves of
involved in both phases. E. litoris, timber of Acronychia pedunculata, bark of E. belahe ballion,
In this study, indomethacin was used as the positive control at a stem barks of Dutaillyea drepacea, leaves of Melicope indica, wood of
dose of 5 mg/kg b. w for the comparison of anti-oedema effect. Esenbeckia species, root bark and aerial parts of Evodia lunu-ankenda
Indomethacin, a cyclooxygenase inhibitor, exerted the maximum anti- (Lal et al. (2005). Further, Lal et al. (2005), isolated evolitrine from
oedema effect during the second phase of oedema due to the reduction dichloromethane extract of twigs of E. lunu-ankenda and acute anti-in-
of prostaglandins which are the second phase mediators. Further, it has flammatory activity was evaluated on carrageenan-induced rat hind
been reported that the second phase of oedema is sensitive to most paw oedema model by only measuring the paw volume at 3rd h after the
clinically effective anti-inflammatory agents (Della et al., 1968). It is injection of carrageenan. As they have shown, evolitrine reduced the rat
interesting to note that all tested fractions/compounds (AELA, EELA, paw oedema in a dose-dependent manner and showed the maximum
AF-EELA and evolitrine) showed maximum inhibition of oedema at the percentage inhibition (78%) at the 3rd hour for the highest tested dose
fifth hour as did the indomethacin. These results may provide some of 60 mg/kg b. w. Although we have measured the paw volume of rats

8
W.M.K.M. Ratnayake, et al. Journal of Ethnopharmacology 238 (2019) 111827

at 1 h intervals up to 5 h, our results at 3rd hour were comparable with adverse effects mainly pro-inflammatory actions (Antosova et al.,
the reported anti-inflammatory activity of evolitrine by Lal et al. 2012). Among the different types of nitric oxide synthase (NOS), Type II
(2005). In the present study, 50 mg/kg b. w. of evolitrine showed 62% or inducible NOS is the major type of NOS which plays a significant role
paw oedema inhibition at 3rd hour. in inflammation. The iNOS which present mainly in macrophages and
In addition to the anti-inflammatory activity, the peripheral an- endothelial cells produce NO that has an important role in various in-
algesic activity was also evaluated using the acetic acid-induced wri- flammatory processes and the inhibition of NO can also be identified as
thing test. Acetic acid is responsible for an increased level of pros- one of the anti-inflammatory mechanisms. Hence, significant
taglandin-E2, prostaglandin-F2, serotonin and histamine in peritoneal (p < 0.05) inhibition of NO level also may contribute to the anti-in-
fluid, hence causing inflammatory pain by inducing capillary perme- flammatory activity of EELA which was clearly observed in this study.
ability (Adeapo and Orherhe, 2013). Further local peritoneal receptors As all the above activities i.e. anti-histamine, anti-oxidant, lipid
are postulated to be partly involved in the abdominal constriction re- peroxidation and NO scavenging, contribute to the anti-inflammatory
sponse. The percentage of inhibition on the number of writhes obtained activity, these findings suggest possible modes of action to explain the
with an effective dose of EELA and evolitrine indicate that they possess anti-inflammatory action of tested extracts and the isolated compound,
peripheral analgesic effect. evolitrine. The prostaglandin E2 (PGE2) and cytokines such as tumor
The anti-nociceptive activity of evolitrine was comparable with re- necrosis factor-α (TNF-α), interleukin-1 (IL-1) and interleukin-6 (IL-6),
ference drug acetylsalicylic acid. The acetylsalicylic acid can inhibit which are pro-inflammatory mediators, contribute to induction of in-
cyclo-oxygenase in peritoneal tissues thus interfering with mechanism flammation and pain by acting on specific ion channels and receptors
transduction in primary afferent nociceptors. The responsive results of (Wang et al., 2014). Previous studies (Fahmy et al., 2017; El Shoubaky
acetic acid-induced writhing suggest the mechanism of evolitrine may et al., 2016; Al-Sayed and Abdel-Daim, 2018) have shown that there
be linked partly to the blockade or release of endogenous inflammatory were significant increases (p < 0.001) in the levels of PGE2, TNF-α, IL-
substances. 1 and IL-6 in the carrageenan treated animals compared to the normal
Histamine is one of the most common and powerful inflammatory groups. Eldahshan and Abdel-Daim (2015) also showed that there were
mediators in the first phase of the carrageenan-induced paw oedema increased levels of PGE2 and TNF-α in paw tissue exudates and PGE2,
model. It causes symptoms of allergic reactions that are mostly involved TNF-α, IL-1 and IL-6 levels in plasma that had been collected after 5 h of
in acute inflammation mediated by the histamine H1 receptor. The 1% carrageenan injection.
histamine H1 receptor mainly contributes to vasodilation, increased of The tested crude extract of A. pedunculata leaves and isolated
vascular permeability and pain at the cellular level (Yong et al., 2013). compound, evolitrine, had marked anti-inflammatory and anti-noci-
As histamine is one of the mediators involved in the initial phase of ceptive activities as shown by a reduction of the oedema in carrageenan
carrageenan-induced oedema, the anti-histamine activity could con- injected paws and the number of writhes induced by 0.6% acetic acid.
tribute to the impairment of the early phase of carrageenan induced As the role of pro-inflammatory cytokines in inflammatory model has
paw oedema. Hence, the observed low anti-histamine activity of EELA been well clarified in many previous studies, in order to better under-
and evolitrine may still contribute to their anti-inflammatory activity. stand the anti-inflammatory and anti-nociceptive mechanisms of tested
The oxygen free radicals can be generated enzymatically during the crude extract and evolitrine, it is important to study their effects on
later phase, by migrated leukocytes, macrophages and damaged en- these inflammatory mediators. The studies on crude EEAL have shown
dothelial cells at the site of inflammation (Boughton-Smith et al., 1993). that it causes a significant (P < 0.05) reduction in plasma PGE2 level in
These play a major role in virtually every step of the development of the adjuvant induced arthritis rat model (Ratnayake, 2018). As PGE2 is
inflammation. Vascular permeability, cellular infiltration and tissue responsible for the oedema formation and also for the pain that ac-
damage can be increased by oxygen free radicals (Boughton-Smith companies the inflammatory reactions, the anti-inflammatory and anti-
et al., 1993; Guzik et al., 2003) which play a key role in the regulation nociceptive effect of A. pedunculate leaves could be attributed in part to
of immune responses (Reddy and Urooj, 2013). The plant extracts the inhibition of PGE2 (Eldahshan and Abdel-Daim, 2015; Williams and
which are having free radical scavenging properties, act as anti-oxi- Peck, 1977) synthesis.
dants and thereby act as anti-inflammatory agents. In the present study, Although cytokines are not constitutively produced under normal
antioxidant activity of EELA was evaluated using in-vitro DPPH free physiological conditions, the inflammatory stimuli induce the gene
radical scavenging activity and in-vivo lipid peroxidation assay to get expression of cytokines. Further, there is evidence that free radicals
more insight into the mechanism of action. As shown in the results could activate oxidative stress sensitive transcription factors, such as
EELA has significant (p < 0.05) in-vitro free radical scavenging ac- nuclear factor kB (NF-kB) (Verri et al., 2012). The NF-kB is known as a
tivity. This indicates that the EELA possesses significant anti-oxidant pro-inflammatory transcription factor that induces the production of
effect, which may have contributed to the inhibition of inflammation COX-2, cytokines, and other pro-inflammatory molecules. As our tested
during the late phase. This activity may be mediated by compounds crude extract showed significant (p < 0.05) in-vitro and in-vivo anti-
such as flavonoids, tannins and polyphenols present in the EELA, as oxidant activity, it could have inhibitory action on production of those
revealed by the phytochemical screening of the extract. pro-inflammatory mediators which could contributed to its anti-in-
Further, reactive oxygen species induced lipid peroxidative tissue flammatory action. Hence, further studies on cytokine levels of in-vivo
damage has been implicated in the pathogenesis of various chronic models are recommended in order to investigate the detailed anti-in-
inflammatory diseases including arthritis (John and Shobana, 2012). flammatory and analgesic mechanisms of the extract of A. pedunculata
Lipid peroxidation is assessed indirectly by the measurement of the and evolitrine.
secondary products such as malondialdehyde (MDA) which is a spon- In acute toxicity study, none of the rats showed mortality or body
taneous breakdown product of peroxides that can be produced from the weight reduction during the study period. According to Allan et al.
free radical attack on polyunsaturated fatty acids (Tukozkan et al., (2012), toxic doses generally cause 10% or more reduction in body
2006). Our study reports the reduction of MDA level in rat serum by the weight. Hence, an absence of body weight reduction in EELA treated
treatment of EELA, indicating that it contains significant (p < 0.05) in- group, indicates the non-toxic nature of A. pedunculata leaf extracts at
vivo anti-oxidant activity. This results further confirmed the antioxidant the tested doses. Further, EELA may not have adversely affected normal
effect, which may have probably contributed to the inhibition of in- metabolism. Besides, there was no significant difference (p > 0.05) in
flammation during the late phase. food consumption between EELA treated and healthy control groups.
Although nitric oxide (NO) acts as an important physiological The average food consumption was constant throughout the study
messenger and effector molecule in many biological systems where it is period in all rats. As food consumption also is one of the most sensitive
present in low concentrations, high concentrations of NO causes indicators of toxicity (Bhardwaj and Gupta, 2012), the comparability of

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W.M.K.M. Ratnayake, et al. Journal of Ethnopharmacology 238 (2019) 111827

food consumption in control and treated groups provide evidence for of oxygen radicals and arachidonic acid and metabolites in the reverse passive arthus
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Authors’ contribution inimbine isolated from Murraya koenigii. Drug Des. Dev. Ther. 11, 103–121.
Jagetia, G.C., Rao, S.K., Balinga, M.S., Babu, K.S., 2004. The evaluation of nitric oxide
scavenging activity of certain herbal formulation in vitro: a preliminary study.
Experimental work, analysis and interpretation of data were carried Phytother Res. 18, 561–565.
out by WMKMR. UGC, AMA, NS and STS contributed through con- Jayaweera, D.M.A., 1982. Medicinal Plants (Indigenous and Exotic) Used in Ceylon Part
V. The National Science Council of Sri Lanka, M. D. Gunasena & Company (Printers)
ception and designing of the project, data interpretation, analysis and Ltd, pp. 2–3.
supervised the study. WMKMR, UGC, AMA, STS and NS contributed John, N.A.A., Shobana, G., 2012. Anti-inflammatory activity of Talinum fruticosum L. on
through drafting and revising of the manuscript. All authors read and formalin induced paw edema in albino rats. J. Appl. Pharm. Sci. 2 (1), 123–127.
Kouloura, E., Halabalaki, M., Lallemand, M.C., Nam, S., Jove, R., Litaudon, M., Awang,
approved the final manuscript. K., Hadi, H.A., Skaltsounis, A.L., 2012. Cytotoxic prenylated acetophenone diamers
from Acronychia pedunculata. J. Nat. Prod. 75, 1270–1276.
Acknowledgment Kozaki, S., Takenaka, Y., Mizushina, Y., Yamaura, T., Tanahashi, T., 2014. Three acet-
ophenones from Acronychia pedunculata. J. Nat. Med. 68 (2), 421–426.
Kumar, V., Abbas, A.K., Fausto, N., Mitchell, R.N., 2007. Robbins Basic Pathology, vol. 8.
The authors are grateful to World Class University Project grant (Ph. Elsevier, pp. 35.
D./15/2012), University of Sri Jayewardenepura, Gangodawila, Kumari, K.D.K.P., Weerakoon, T.C.S., Handunnetti, S.M., Samarasinghe, K., Suresh, T.S.,
2014. Anti-inflammatory activity of dried flower extracts of Aegle marmelos in
Nugegoda, Sri Lanka for financial support.
Wistar rats. J. Ethnopharmacol. 151, 1202–1208.
Kuo, C.L., Chi, C.W., Liu, T.Y., 2004. The anti-inflammatory potential of berberine in vitro
Appendix A. Supplementary data and in vivo. Cancer Lett. 203, 127–137.
Lal, B., Bhise, N.B., Gidwani, R.M., Lakdawala, A.D., Joshi, K., Patvardhan, S., 2005.
Isolation, synthesis and biological activity of evolitrine and analogs. Arkivoc 2,
Supplementary data to this article can be found online at https:// 77–97.
doi.org/10.1016/j.jep.2019.111827. Lesueur, D., Serra, D., De, R., Bighelli, A., Hoi, T.M., Thai, T.H., Casanova, J., 2008.
Composition and anti-microbial activity of the essential oil of Acronychia peduncu-
lata (L.) Miq. From Vietnam. Nat. Prod. Res. 22 (5), 393–398.
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