Synergistic Antinociceptive Activity of Combined Aqueous Extracts of Artemisia Campestris and Artemisia Herba-Alba in Several Acute Pain Models

SUPPLEMENTARY MATERIALS

Synergistic antinociceptive activity of combined aqueous extracts of Artemisia campestris and Artemisia herba-alba in several acute pain models

Imed Eddine Kadi, Mohamed Ouinten, Nadhir Gourine*, Mohamed Yousfi

Laboratory of Fundamental Sciences. University of Amar Telidji, Road of Ghardaia, Po.Box 37G (03000), Laghouat, Algeria

*Corresponding author at:

Laboratoire LSF, B.P. 37G, Université Amar TÉLIDJI, Laghouat, (03000), Algeria.

Tel.: +213 29 90 00 66

Fax: +213 29 90 00 66

E-mail address: [email protected] [email protected]

Abstract: In this study, total phenolic and flavonoid contents, acute toxicity and the antinociceptive activity of Artemisia campestris and Artemisia herba-alba individually and in combination were investigated using multiple forms of pain in animals. Our results have been shown that plants are relatively safe without clinical signs of toxicity in animals. Thus, extracts were presented high levels in phenolic and flavonoid contents. Artemisia decoctions with 100, 200, 400 mg/kg b-w studied dose, clearly attenuate chemical and thermal noxious stimuli in writhing, formalin and hote plate tests, and significantly reduced paw edema in formalin test. Additionally, binary combination forms exhibited a great improvement in intensity and amplitude of antinociceptive activity in comparison with both plants used individually by a relative interference with opioid system. Our findings suggested the central and peripheral analgesic properties and confirmed the folkloric medicinal use of these plants in pain symptom treatment.

Key words: Antinociceptive, Analgesic, Artemisia campestris, Artemisia herba-alba, Pain, Nociception.

1 1. Complementary introduction

Somatic and mental health problems were often accompanied with a pain syndrome. It’s a major symptom in most chronic and acute diseases, especially those related to locomotor system (Dillworth et al. 2012, Marchand 2014). Pain is a complex phenomenon implicated neuro-physiological pathways and neuro-chemical mediators from peripheral nociceptors to higher centers in brain (Sinatra et al. 2009, Motoc et al. 2010). However, their adverse effects are unbearable in patients. It’s widely documented that NSAIDs generate cardiovascular effects, gastrointestinal ulcers and renal insufficiency (Higgs et al. 2013, Ymele et al. 2013). In addition, the prolonged use of opioids induces a tolerance and hyperalgesie. Which require us to research an alternative healthy and effective biopharmaceuticals.

Natural products imposed themselves for a long time as an important and Inexhaustible origin in new therapeutics research. After the discovery of Artemisinin an anti-malarial agent from Artemisia annua, a great pharmacological interest was brought to Artemisia genus (Marchand 2014). Although artemisinin was found to be the major metabolite for its antimalarial effects, several flavonoids and terpenoids are considered to possess biological activities when used alone and also to synergistically boost the bioavailability of artemisinin. However, due to the limited quantities of these metabolites in wild plants, in vitro cultures were established and strategies have been adopted to enhance medicinally important secondary metabolites in these cultures.(Ali et al. 2017)

Grouping over 400 species with a wide distribution along the Mediterranean cost, Artemisia genus was considered to be a great source of biomolecules (Sainz et al. , Valant-Vetschera et al. 2003).

In recent years (since 2010) the extracts of Artemisia plants were extensively studied, leading to isolation of 159 compounds with diverse structures from this genus (Zhang et al. 2017). The studies conducted in these plants (mostly based in vitro experiments) have revealed their potential in anti-inflammation, anti-cancer, antimalarial, antinociception and antimicrobial and antimicrobial. (Zhang, et al. 2017). More importantly, Hispidulin (40, 5, 7-trihydroxy-6- methoxyflavone) is a flavones derivative found in Artemisia species among other ones which have antioxidant, antifungal, anti-inflammatory, antimutagenic, and antineoplastic properties (Patel and Patel 2017).

Artemisia herba-alba is a spontaneous aromatic plant frequently used in Algerian and Tunisian traditional medicine (Qnais et al. 2014, Aidi Wannes and Marzouk 2016, Najjaa et

2 al. 2017) to relieve gastric disorders with antispasmodic, hypoglycemiant, anti-inflammatory properties (Lee et al. 2012), vasorelaxant (Naoufel et al. 2016) and diabetes treatment (Aidi Wannes and Marzouk 2016, Telli et al. 2016, Bushara et al. 2017, El-Marasy et al. 2017). Similarly, the use of Artemisia campestris in Algerian folkloric medicine is recommended for its anti-Rheumatic, anti-inflammatory, diabetes treatment and anticancer activities (Naskar et al. 2013, Barkat et al. 2015, Aidi Wannes and Marzouk 2016). The meeting point of both previous plants it’s their extensively use in the management of several forms of abdominal (Silva et al. 2002), gastric (Wagner and Ulrich-Merzenich 2009) and Rheumatic (Motoc, et al. 2010) pain paradigms.

The strategy of natural products combination is an effective approach to optimize and improve pharmacological and therapeutic properties of phytopharmaceuticals in order to treat multifactorial diseases (Wagner and Ulrich-Merzenich 2009). It has been frequently reported that herbal extracts in combination improve the bioavailability (Yang et al. 2014), neutralize the side effects of ingredients and optimize their pharmacokinetics and pharmacodynamics properties (Williamson 2001, Wagner and Ulrich-Merzenich 2009).

2. General literature background

Antinociceptive (Qnais, et al. 2014) and anti-inflammatory (Khlifi et al. 2013) activities of A. herba-alba, and anti-inflammatory properties in vivo for A. campestris (Fotso et al. 2014, Ghlissi et al. 2016) were previously confirmed.

Moreover, it’s widely documented that phenolic compounds, in particular flavonoids plays an effective role in nociceptive response management (Mothana et al. 2012, Higgs, et al. 2013, Wang et al. 2014, Rauf et al. 2016). Detailed studies reported the phenolic molecular profile of these Artemisia plants; flavonols and flavonones are the main categories (Sefi et al. 2010, Megdiche-Ksouri et al. 2015, Dif et al. 2016, Bourgou et al. 2017), with phenolic acids, coumarine and its derivative in A. campestris (Ferchichi et al. 2006, Dib et al. 2016). In addition, sesquiterpene lactones, flavonoids from flavone and flavonol glucosides to highly methylated flavonoids and phenolic acids such as chlorogenic acid with their derivatives have been identified in A. herba-alba (Mohamed et al. 2010, Bourgou, et al. 2017, Peron et al. 2017). Furthermore, Hispidulin (40, 5, 7-trihydroxy-6-methoxyflavone) is a flavone derivative found in both Artemisia herba-alba and campestris exhibited antioxidant, antifungal, anti- inflammatory, antimutagenic, and antineoplastic properties (Patel and Patel 2017). Some very

3 recent publications reported the antioxidant and the biological activities of the total phenolic compounds of Algerian A. herba alba and A. campestris plants using several solvents extractions: organic (methanolic, ethanolic, hexanic) or aqueous, (Dif, et al. 2016, Belkacem et al. 2017, Dib et al. 2017, Salhi, Rahmani, et al. 2017, Salhi, Saghir, et al. 2017).

This variability in the qualitative and quantitative levels on molecular profile stimulated us to mix extracts from these plants to hone their analgesic properties. Acetic acid, Formalin and Hot-plate tests using mice and rats were applied in this study.

Writhing test is a non-selective classical method to evaluate the antinociceptive responses (Silva et al. 2015). Acetic acid injected in animals abdomen was induced an irritation process (Zakaria et al. 2008), accompanied with an increase in endogenous mediator levels in abdominal cavity such as histamine, serotonin, bradykinin, substance P and prostaglandin (Reanmongkol et al. 2009), especially PGE2 and PGF2 (Ymele, et al. 2013). Thus, the release of pro-inflammatory cytokines IL1, IL6, IL8 and TNFα (Silva, et al. 2015) with increasing in capillary permeability. All the preceding molecules were participated in the stimulation of peripheral and central chemo-sensitive receptors (Bukhari et al. 2010). Aqueous extracts gavages individually and in combination, showed a remarkable antinociceptive effect in writhing test by immediate effects on prostaglandin actions, or indirectly by interference with acid arachidonic metabolism and inhibiting of cyclooxygenase and lipooxygenase functions (Zakaria, et al. 2008, Fotso, et al. 2014).

Intra-plantar injection of formalin in rats was generated a moderate continuous pain sensation (Reanmongkol, et al. 2009) distributed in two forms, chronologically and neuo- physiologically different (Nonato et al. 2011). Neurologic pains (Early phase 0-10 min) were created by a direct stimulation of sensory neurons such as C fibers, and inflammatory pains (Late phase 15-60 min) were discussed by increasing in inflammatory mediators such as prostaglandins, serotonin, histamine and bradykinine (Bukhari, et al. 2010). A quiescent- interval period separates previous phases (Silva, et al. 2015). Peripheral analgesic drugs such as NSAIDs, Diclofenac and aspirin are known as inhibitors of inflammatory nociception, whereas opioids central drugs can attenuate both forms of pain (Ymele, et al. 2013).

Hot-plate test is a selective model to appreciate the central analgesic effects by direct stimulation of nociceptors C and Aδ (Reanmongkol, et al. 2009), characterized by the involvement of central neural structures such as spinal cord and cortical centers (Silva, et al. 2015).

4 Combination therapy is one of the most efficient approach in pain syndrome management (Fotso, et al. 2014). Accordingly, several studies in literature showed a synergy between various herbal extracts in order to perfect the analgesic activities. Su et al. (Su et al. 2012) have reported that combined extracts from Commiphora myrrha and Bosuellia carteri were exerted a profound analgesic effect as those found in individual treatments. Similarly, Kumari et al. (Kumari et al. 2015) valorized chloroform extracts combination from Lawsonia inermis and Chlorophytum borivilianum, as a potent analgesic extract.

3. Experimental

3.1. Animals

Healthy Swiss albino male mice 20-30g and wistar male Rats 150-200g were obtained from animal unit of Pasteur institute of Algiers and Pharmacy department of Constantine III university. Mice and Rats were directly acclimatized in favorable conditions during 10 days for adaptation. They were fed with standard food (ONEB, Béjaia, Algeria) and water ad- libitum throughout the period of study. Animals were fasted overnight with free access to water before each analgesic test.

3.2. Vegetal material and extraction procedure

3.2.1 Plants brief descriptions

Artemisia campestris is a shrub of 40-80 cm, reddish erect stem, Dark green glabrous leaves. The flowers are small heads, conical, yellowish green (Chehma 2006). Artemisia herba alba (asso) is an under shrub of 20-40cm, whitish dry appearance, leaves divided into fine woolly and flowers grouped in clusters with very small and ovoid heads (Chehma 2006, Bezza et al. 2010). 3.2.2 Plants phonological stages For Artemisia herba-alba (asso), the autumn season is considered as the favorable period for vegetative growth. Flowering begins in June and develops essentially in late summer. The shoots that originate from the lateral buds of the base of lignified branches appear in winters. During the summer, the plant reduces these leaves, one of the most effective morphological adaptations. An indication of adaptation to the dryness is provided by its root as well in its form, mode of extension and its biomass (Ghrabi 2005).

5 Observations of the phonological events for Artemisia campestris showed that bud break occurred during the period from mid to late February month. Flowering of the plant begin in August and continued up to the end of November. 3.2.3 Plants collection Spontaneous A. herba-alba and A. campestris aerial parts were collected in September 2015 from Laghouat (Aflou) region in south Algeria. Plant samples were set as a mixture of four different populations collected. Sampling was done by cutting the outer two thirds of each plant by hand without any effect on the root system. The collected samples are conserved in paper bag in a dry place and protected from light. These plants were identified in fundamental sciences laboratory by Professor Mohamed OUINTEN. The different plant samples were collected during the flowering stage; (Mid September for both A. herba-alba and A. campestris). Voucher specimens (AHA-AFL/09/15 and AC-AFL/09/15, respectively) were deposited in the herbarium of the Fundamental Sciences Research Laboratory at Laghouat University. Plants were immediately dried from the sun’s rays and any sources of energy during two weeks. Aerial part of each plant (which consists of stems, leaves and flowers) was powdered to fine particles. Aqueous extracts were prepared by decoction procedure. 200g of each plant was mixed in 1L of distilled water and the mixture was put in water path at 80°C for 1h. Both aqueous extracts AEAC and AEAHA were filtered through Whatman’s filter paper and concentrated on rotavapor apparatus at 45°C. Finally, concentrated extracts were conserved in refrigerator for future use (Mohamed, et al. 2010).

3.3. Chemicals

Folin-ciocalteau reagent, Sodium carbonate, Gallic acid, Aluminum chloride and Quercetin were purchased from Sigma chemical Inc. (St. Louis, MO, USA). All other chemicals such as Aspirin, Morphine and Naloxone were purchased from local Pharmaceutical industry.

6 4. Experimental procedure

4.1. Total polyphenols quantification

Total phenolic content of extracts were estimated using the classical method of Folin- ciocalteau reagent described by Megdiche et al. (Megdiche-Ksouri, et al. 2015). 125µL of each samples were mixed with 125µL of Folin-ciocalteau phenol reagent. 500µL of distilled water were added to mixture. After stirring, 1250µL of Na2CO3 were added and followed by incubation in dark room for 1.5h at ambient temperature. In parallel, a calibration curve (10- 200µg/mL) of gallic acid used as standard was prepared. The absorbance reading of various tests is carried out at 760 nm using spectrophotometer. The total phenolic of both Artemisia aerial parts were expressed as mg acid gallic equivalents /g of extracts (mg GAE/g extract). Three repetitions for all samples analysis were performed.

4.2. Total flavonoids quantification

Flavonoids contents were determined by AlCl3 colorimetric analysis described by Quettier De

Leu et al. (Quettier-Deleu et al. 2000). 1mL of each extract was added to 1mL AlCl3.6H2O (2%), the mixture was incubated in a dark room for 1 hour. A series of different concentration (5, 10, 50, 100, 150 and 200 µg/mL) were prepared from Quercetin solution used as standard. Mixtures absorbance was determined in 430 nm by spectrophotometer and the results were expressed as mg EQ/g of extract. All samples were analyzed in triplicate.

4.3. Acute Toxicity study

Acute toxicity of both A. herba-alba and A. campestris aqueous extracts were studied according to model described by Sayed Mohamed et al. (Mohamed, et al. 2010). Animals were distributed to four groups for each plant, and treated intraperitoneally by different doses 500, 1000, 1500. 2000 mg/kg b-w. All changes in animals’ behavior or appearance of clinical signs of toxicity were recorded in first 4 hours, and the number of deaths was counted at 24h after treatments.

7 4.4. Acetic acid induced abdominal stretching in mice

Abdominal acute pain model was studied by writhing test. 66 male mice were distributed (n=6) to 5 lots, the first contains negative control animals orally pretreated with saline (0.9%; 10mL/kg). Lot II for positive control, animals were orally pretreated by aspirin 200 mg b-w. Lot III contains 3 groups, mice were pretreated by gavages of different doses of A. campestris aqueous extracts. 100 (group III), 200 (group IV), 400 mg/kg (group V). Lot IV collects mice of different pretreatment with A. herba-alba aqueous extracts 100 (group VI), 200 (group VII), 400 mg/kg (group VIII). The last lot was oriented for combination study between previous extracts, it’s contains 3 groups (IX, X, XI). Animals were orally pretreated with AC 400 mg/AHA 400 mg with different ratios 50/50, 75/25, 25/75 respectively. After 30 min of all experimental treatments mentioned above, 1% (v/v) (10mL/kg) of acetic acid was injected i.pin each animal isolated individually in a large glass cylinder. The number of abdominal constrictions was recorded for a period of 30 min after chemical nociceptive stimulation and inhibition percent were determined by comparison of all groups results with control group (Park et al. 2013).

4.5. Formalin induced neurological and inflammatory pain in Rats

According to Lee et al. (Lee, et al. 2012) Neurologic and inflammatory pain models were studied using Formalin test. Males Rats were divided to 11 groups with 6 Rats each. Group I for negative control contains animals pretreated with saline (0.9%, 10mL/kg). The second, group II represent a positive control with Diclofenac pretreatment at the dose 50 mg/kg b-w. for A. campestris evaluation, three groups were used with different doses 100 (group III), 200 (group IV), and 400 mg/kg (group V). The following groups VI, VII, VIII were used for A. herba-alba study at doses 100, 200, 400 mg/kg b-w respectively. In combination experiment, three different reports were determined from AC 200 / AHA 400 combination. 50/50 (group IX), 75/25 (group X), 25/75(group XI).

All doses of saline, Diclofenac and extracts were administered by gavage one hour before pain induction. Each animal was individually isolated in color plastic cage (22×30×15cm) and 50µl of formalin (5%) was intraplantar injected to generate an inflammatory process in right paw. All changes in animal behavior were observed and the time of paw licking and lifting was measured using a chronometer in 5min intervals for 1h under a strict conditions.

8 4.5.1. Paw edema evaluation

Intraplantar injection of (50µL, 5%) Formalin in dorsal left paw generated a pain syndrome with an inflammatory process (Edema). The Paw thickness was measured before and one hour after formalin injection by a fin micrometer. The percent of paw edema increasing was calculated as a report of paw thickness before and after formalin injection.

4.6. Thermally induced pain model

The central analgesic properties were studied using a physical origin in pain induction (Naskar, et al. 2013). Selected mice were divided into different groups (n=6). Control mice (group I) were treated i.p with Morphin 10mg/kg b-w. Three groups for A. campestris doses II, III, IV, were treated orally by aqueous extracts 100, 200, 400 mg/kg b-w respectively. The same doses were also administered in A. herba-alba groups, 100 (group V), 200 (group VI), 400 mg/kg (group VII). Other three groups were reserved for the combination test by selecting the effective dose of both Artemisia plants 400mg/kg b-w. Three reports were selected, 50/50 (group VII), 75/25 (group VIII), 25/75 (group IX). Each animal served as its own control. After 30 min of all treatments, each animal was placed individually in glass beaker on hot plate (55±1°C) and the latency of discomfort reaction (licking, jumping, lifting) was recorded in (s) during 3h (Sheikh et al. 2016). The cuttoff time used to prevent skin damage was 25sec.To estimate the involvement of opioids receptors in antinociceptive response, Naloxone an antagonist of these receptors was used.

5. Statistical analysis

All results were expressed as mean ± “standard error of mean” (SEM). The statistical study was carried out by analysis of variance (ANOVA) followed by Tukey’s Post-hoc test, using Graphpad prism statistical program (software, version 6.0). Significance levels was set at p<0.05.

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Figure S1: Aqueous extracts from Artemisia campestris (A), Artemisia herbaalba (B) and binary combination forms (C) attenuate Acetic acid induced visceral pain in animals. Results

10 are mean ± SEM, 6 repetition for all groups. One way ANOVA analysis followed by Tukey’s Post-hoc test. *p˂0.05, **p˂0.001, ****p˂0.001 in

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Figure S2: Artemisia campestris aqueous extracts attenuates neurologic (B) and inflammatory (C) pain models. Thus paw edema (D) in male Rats. Results were expressed as mean±SEM (n=6), statistical analysis was applied with ANOVA followed by Tukey’s post- hoc test (*p<0.05; **p<0.01; ****p<0.001) in comparison with control group.

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Figure S3: Artemisia herba-alba aqueous extracts attenuates neurologic (B) and inflammatory (C) pain models. Thus paw edema (D) in male Rats. Results were expressed as mean±SEM (n=6), statistical analysis was applied with ANOVA followed by Tukey’s post- hoc test (*p<0.05; **p<0.01; ****p<0.001) in comparison with control group.

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Figure S4: Combination of Artemisia campestris and Artemisia herba-alba aqueous extracts attenuates neurologic (B) and inflammatory (C) pain models. Thus paw edema (D) in male Rats. Results were expressed as mean±SEM (n=6), statistical analysis was applied with ANOVA followed by Tukey’s post-hoc test (*p<0.05; **p<0.01; ****p<0.001)in comparison with control group

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