Vincetoxicum arnottianum ameliorate inflammation by suppressing oxidative stress and pro-inflammatory mediators in rat

Journal Pre-proof Vincetoxicum arnottianum ameliorate inflammation by suppressing oxidative stress and pro-inflammatory mediators in rat Zartash Zahra, Muhammad Rashid Khan, Sayed Afzal Shah, Sonia Maryam, Muhammad Majid, Tahira Younis, Moniba Sajid PII:

S0378-8741(19)33424-5

DOI:

https://doi.org/10.1016/j.jep.2020.112565

Reference:

JEP 112565

To appear in:

Journal of Ethnopharmacology

Received Date: 29 August 2019 Revised Date:

1 January 2020

Accepted Date: 8 January 2020

Please cite this article as: Zahra, Z., Khan, M.R., Shah, S.A., Maryam, S., Majid, M., Younis, T., Sajid, M., Vincetoxicum arnottianum ameliorate inflammation by suppressing oxidative stress and proinflammatory mediators in rat, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/ j.jep.2020.112565. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

Fractionation MeOH • • • •

Drying Grinding Extraction Filtration

• • • • •

Protection from heat induced denaturation of protein

Methanol (VAM) n-hexane (VAH) Ethyl acetate (VAE) n-butanol (VAB) Aqueous (VAA)

VAE

Rutin, Gallic Acid, Caffeic Acid, Apigenin And Myricetin

Vincetoxicum arnottianum

Carrageenan Formaldehyde

Carrageenan and formaldehyde induced paw edema in rat

VAM VAE Inflammation, trauma

Freund's adjuvantinduced arthritis

Paw volume Body weight Spontaneous activity CAT, SOD,POD

HPLC-DAD analysis

Vincetoxicum arnottianum ameliorate inflammation by suppressing oxidative stress and pro-inflammatory mediators in rat Zartash Zahraa, Muhammad Rashid Khana,*, Sayed Afzal Shahb, Sonia Maryama, Muhammad Majidc, Tahira Younisa,d, Moniba Sajida a

Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University,

Islamabad, Pakistan b

Department of Plant Sciences, Faculty of Biological Sciences, Quaid-i-Azam University,

Islamabad, Pakistan c

Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University,

Islamabad, Pakistan d

Faculty of Life Sciences, Department of Zoology, Government College University

Faisalabad, Faisalabad, Pakistan *Corresponding author

Email address [email protected] (Z. Zahra) [email protected] (M. Rashid Khan) [email protected] (S. Afzal Shah) [email protected] (S. Maryam) [email protected] (M. Majid) [email protected] (T. Younis) [email protected] (M. Sajid)

Abbreviations ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase; CAT, catalase; FCA, Freund’s complete adjuvant arthritis; POD, peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase; TFC, total flavonoid content; TPC, total phenolic content;

Abstract Ethnopharmacological relevance: Aerial parts of Vincetoxicum arnottianum (Wight) Wight (Family Apocynaceae) are used by local communities for inflammation, healing of wound and injuries and also for urticaria. Aim of study: Extract/fractions of V. arnottianum were evaluated for potential antiinflammatory activity in rat. Methods: Methanol extract of aerial parts of V. arnottianum (VAM) was partitioned on polarity for n-hexane (VAH), ethyl acetate (VAE), butanol (VAB) and aqueous (VAA) fractions. The extract/fractions were evaluated during in vitro assay for protection against heat induced protein denaturation and Carrageenan induced paw inflammation in rat. VAM and VAE were evaluated for anti-inflammatory potential against formalin and Freund’s complete adjuvant (FCA) induced inflammation in paw of rat while croton oil induced inflammation in ear of rat, respectively. The level of inflammatory mediators; IL-17, IL-1β, IL-6, TNF-α and nitric oxide (NO) was estimated in serum of rat. Results: All the extract/fractions used in this study exhibited anti-inflammatory activity. However, VAE (300 mg/kg) exhibited potential anti-inflammatory activity in carrageenan (78.06±4.6%), formalin (54.71±0.34%) and croton oil (73.12±1.9%) induced edema in rat. In FCA induced inflammation model VAM and VAE showed admiring proficiencies against alteration of body weight and organ weight indices, paw edema and histological studies. In serum increased level of pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-17) and NO during adjuvant-induced inflammation were more efficiently restored with VAE treatment to rat. Presence of polyphenolics; rutin, gallic acid, caffeic acid, apigenin, myricetin and quercetin was indicated in VAE. Conclusion: The results suggest the presence of anti-inflammatory constituents in V. arnottianum. Key words: Vincetoxicum arnottianum; Antioxidant; anti-inflammatory; Freund’s complete adjuvant inflammation 1. Introduction Since ancient times inflammatory disorders have been treated with plants and plant derived formulations (Sajid et al., 2017; Maryam et al., 2019). Vincetoxicum arnottianum (Wight) Wight is a perennial undershrub and is distributed in different areas of Pakistan covering Swat and Hazara districts of Khyber Pakhtunkhwa and also in Kashmir. It is characterized by purple flowers with bearded corolla and rhomboid corona. Vincetoxicum arnottianum (syn. Vincetoxicum hirundinaria; Vincetoxicum officinale; Cynanchum arnottianum) is locally

used for the treatment of injuries, wounds, swelling and bruising (Beigh et al., 2004; Zaidi and Crow, 2005; Khanum et al., 2013; Majid et al., 2019). It has been reported that urticaria (a skin disease) is treated with juice of V. arnottianum leaves in northern areas of Pakistan (Shah et al., 2016). Two alkaloids, namely 10 β-(−)-antofine N-oxide and 10 α-(−)-antofine N-oxide were isolated from the stem bark and the root bark of V. arnottianum (Lavault et al., 1999) as well as two new natural products, ( - )-( R )-13a R -secoantofine and ( - )-( R )-13a R -6- O - desmethylsecoantofine (Stærk et al., 2002) which have the ability to cure inflammatory related disorders have been isolated. V. arnottianum along with other species of this genus, are used for ailments like gastritis, malaria, cholera, asthma, skin diseases, ulcer and constipation (Hussain and Ghani, 2008). The plant is also used in various conditions; bruising,

pain,

trauma,

bone

fractures,

asthma

(https://www.medicinetraditions.com/vincetoxicum-swallow-wort.html). V. arnottianum is also used against inflammation related disorders (Engel et al., 2016). Anticancer activities of V. arnottianum against bone and breast cancer cell lines have been reported in earlier studies (Engel et al., 2016). Ethanol extract of Cynanchum paniculatum and C. acutum exhibited potential anti-inflammatory activity (Choi et al., 2006; Estakhar et al., 2012). Presence of polyphenolic and antioxidant activity has been studied for Vincetoxicum lutea (Šliumpaitė et al., 2013). Antidiabetic and antioxidant activities of C. acutum have been studied (Fawzy et al., 2008). Suppression of inflammatory mediators during inflammation was studied for Cynanchum wilfordii (Cho et al., 2017) and C. atratum (Choi et al., 2017).There is no earlier report of the evaluation of V. arnottianum plant in anti-inflammatory activity. On account of its use in various inflammatory disorders and the presence of anti-inflammatory constituents, V. arnottianum extract/fraction was evaluated for its antioxidant and anti-inflammatory activity. 2. Materials and methods 2.1. Extract preparation Aerial parts of V. arnottianum were collected from mountain near village Deri Nala, the surroundings of district Kotli of Azad Jammu and Kashmir, Pakistan. The plant was identified by Dr. Sumaira Sahreen, Associate Curator and a voucher specimen (063521) was deposited at the Pakistan Museum of Natural History, Islamabad, Pakistan. After shade drying 3 kg powder was extracted thrice with 10 liters of commercial methanol for 48 h at 25 °C. Filtrate obtained was dried in rotary vacuum evaporator at 40 ºC to get methanol extract (VAM). From this 50 g of VAM was used for polarity wise fractionation in arrangement of n-

hexane (VAH), ethyl acetate (VAE), butanol (VAB) and soluble aqueous (VAA). The fractions obtained were dried as above and stored at 4 ºC for different studies. 2.2. HPLC analysis For HPLC-DAD analysis of VAE Agilent C-8 system was used. Briefly, acetonitrilemethanol-water-acetic acid (5:10:85:1) was used as mobile phase A while acetonitrilemethanol-acetic acid (40:60:1) as mobile phase B. Initially for 0-20 min a gradient of 0 to 50% B, for 20-25 min a gradient of 50 to 100% B and then isocratic 100% B till 30 min was utilized. Injection volume was 20 µl with flow rate of 1 ml/min. evaluation of rutin and gallic acid was performed at 257 nm, catechin at 279 nm, caffeic acid at 325 nm whereas quercetin, myricetin and kaempferol were analyzed at 368 nm (Zu et al., 2006). The experiment was conducted thrice. 2.3. Studies on albumin denaturation Protection of heat induced albumin denaturation assay was performed for various extract/fractions (Leelaprakash and Dass, 2011). The extract/fractions at various concentrations (100, 200, 300, 4000, 500 µg/ml) were mixed with 1% aqueous solution of bovine albumin and incubated at 37 ºC and then at 51 ºC for 20 min. After cooling the absorbance was noted at 660 nm. The percent inhibition of protein denaturation was calculated as: % ℎ

=











× 100

2.4. Experimental animals In this study Sprague-Dawley male rats having weight of ~150-200 g were housed at 12 h light/dark cycle, 25 ± 1°C temperatures in ordinary cages and were fed with rodent feed and water ad libitum. The guide lines of Quaid-i-Azam University, Islamabad, Pakistan (Bch0328) were followed for experimentation. 2.5. Acute toxicity studies in rat For this purpose Sprague-Dawley male rats were divided in to six groups having 6 animals in each group. After acclimatization animals of Group I was orally administered at 150 mg/kg dose and followed for 15 days to examine the toxicity. The test group did not show sign of toxicity the experiment was followed at higher doses of 500, 1000, 2000 and 3000 mg/kg and examined each rat for 15 days on daily basis. The toxicity studies were made on mortality, body weight and behavioural pattern such as urination, purgation, diarrhea, food intake, piloerection, salivation, lacrimation, nasal secretion, physical activity, mood and aggression.

Toxicity studies were made by following the guidelines 425 of Organization for Economic Cooperation and Development (OECD). 2.6. Anti-inflammatory studies in rat 2.6.1. Carrageenan-induced inflammatory activity in rat Anti-inflammatory potential of V. arnottianum extract/fraction was evaluated by carrageenaninduced hind paw edema model in Sprague-Dawley male rats (Muhammad et al., 2012). There were 13 groups with 7 rats in each group. Animals of Group I (control) were treated orally with 1 ml/kg of 0.9% w/v saline. Group II was treated orally with I ml/kg of 10% DMSO, I h before the injection of 100 µl of carrageenan suspension (0.9% w/v in saline) to right hind paw of rat. Rats of Group III – XII were treated with extract/fractions at 150 mg/kg and 300 mg/kg whereas Group XIII was treated with diclofenac potassium at dose of 10 mg/kg, 1 h earlier to carrageenan treatment. At various time periods (0, 1, 2, 3, 4 h) paw volume was recorded by Plethysmometer. Anti-inflammatory activity was reported as percent inhibition of edema. 2.6.2. Formalin-induced paw edema The rats were randomly divided in to six groups having 3 rats in each group. The edema was induced in the right hind paw of all rats by injecting 100 µl of 2% aqueous solution of formalin. Group I was formalin control (treated with distilled water) whereas Group 11Group V was administered with VAM and VAE at 150 mg/kg and 300 mg/kg body weight. Ibuprofen at dose of 10 mg/kg was administered to Group VI as positive control. The animals were treated 45 min before formalin injection. Anti-inflammatory effects were recorded at 1, 3, 6, 12 and 24 h after formalin injection (Sadeghi et al., 2014). 2.6.3. Croton oil-induced ear edema Sprague Dawley male rats (21) were divided in to seven groups with equal number of rats in each. Group I was normal control and acetone was applied topically on both ears. Edema was induced by applying 100 µl of 5% croton oil (in acetone) on the inner surface of right ear of animals other groups (Group II- Group VII) while acetone was applied on the left ear. Application of VAM (Group III, IV) and VAE (Group V, VI) was made I h before the application of croton oil at a dose of 2.5 mg/ear and 5.0 mg/ear. The rats of positive control (Group VII) were treated with Ibuprofen (1 mg/ear) 1 h before the induction of edema. Rats were sacrificed after 4 h of treatment and weight of a plug measuring 7 mm (diameter) was recorded on both ears of a rat (Reanmongkol et al., 2009). 2.6.4. Freund’s complete adjuvant-induced inflammation in rat

The rats were divided in to seven groups with three rats in each. Group I was normal control and treated with 0.9 % saline (2 ml/kg) while rats of remaining groups were injected with 200 µl from stock of Freund’s complete adjuvant (10 mg of heat-killed Mycobacterium tuberculosis/ml liquid paraffin) subcutaneously at the base of the tail under anaesthesia (Zhang et al., 2009). Among these rats Group II received Freund’s complete adjuvant (FCA) alone. Group III and IV were treated with VAM (150 mg/kg and 300 mg/kg) while Group VVI with VAE (150 mg/kg and 300 mg/kg). Group VII was administered with ibuprofen at 10 mg/kg body weight. Two schemes were followed for anti-arthritis assessment. 2.6.4.1. Treatment mode According to this scheme VAM and VAE was administered to FCA treated rats after 13 days at 150 mg/kg and 300 mg/kg and ibuprofen (10 mg/kg) orally on alternate days and reversal of arthritic symptoms was assessed. 2.6.4.2. Preventive mode In this scheme FCA treated rats were administered with VAM and VAE at 150 mg/kg and 300 mg/kg and ibuprofen at 10 mg/kg) orally on alternate days from the day 1 of FCA treatment. Prevention of arthritis was assessed in comparison to control. 2.6.4.3. Adjuvant-induced inflammation scoring For this a well-established scoring system (Han et al., 2016) was used to record the signs of arthritic severity. The system graded the arthritic severity on five point scores related to swelling, induration and erythema and these were recorded on daily basis. Plethysmometer (Ugo Basile 7140, Italy) was used for measurement of paw volume. 2.6.4.4. Histological investigation The rats were treated by intraperitoneal administration 1% chloralose and 25% urethane at a dose of 1 mg/kg and rats were sacrificed on the last day (25th day) of experiment. The tissue of arthritic joint was fixed in buffered formalin (10%, pH 7.4). After processing the tissue in escalating concentration of ethanol, thin sections (5 µm) were made. Eosin and haematoxylin were used for staining purpose. 2.6.4.5. Studies on body weight and relative organ weight The body weight of each rat was recorded on the last day of experiment and after dissection liver, kidney, spleen and thymus were excised and their weights were recorded. 2.6.4.6. Biochemical analysis of blood Blood sample of each rat was assorted under anaesthesia via the abdominal aorta in BD vacutainer for biochemical studies. From each blood sample serum was separated by centrifuging at 6000 rpm for 15 min at 4 °C and stored at -20 °C.

In the serum the level of protein, urea, creatinine, total bilirubin, aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) were recorded with AMP standard diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria). 2.6.4.7. Studies on antioxidant enzymes in serum In serum of each rat activity of catalase (CAT) was measured on the rate of H2O2 hydrolysis at 240 nm (Sundaram et al., 2015). For the measurement of superoxide dismutase (SOD) and peroxidase (POD) activity in serum an earlier reported method was used (Sajid et al., 2016). The quantity of reactive oxygen species (ROS) in serum was measured according to the earlier described method (Hayashi et al., 2007). 2.6.4.8. Studies on cytokines in serum In the serum of FCA treated rats the level of cytokines; IL-1β and IL-6, TNF-α and IL-17 was measured by enzyme-linked immunosorbent assay (ELISA). For measuring the level of cytokines; IL-1β and IL-6 the kit (BD Biosciences, USA) while for TNF-α and IL-17 ELISA kit of Ray-Biotech, Inc. (Norcross, GA, U.S.A) was used. 2.6.4.9. Measurement of nitric oxide in serum For the measurement of NO level 30 µl of serum was mixed with 0.3 M NaOH and 5% ZnSO4. After centrifugation (6400 ×g) for 15–20 minutes from the supernatant an aliquot of 10 µl was mixed with 200 µl of Griess reagent in 96 well plate. At 540 nm the absorbance of reaction mixture was noted. Dose response curve of sodium nitrite was used nitrite content in serum (Batool et al., 2017). 2.7. Statistical analysis For the quantitative experiments results were expressed as mean ± SD. The data were analyzed by one way analysis of variance and means were compared at P-value ≤ 0.05 for level of significance by Tukey’s multiple comparison test among groups by Statistix 8.1. 3. Results 3.1. Extraction yield The yield of V. arnottianum methanol extract (VAM) obtained was 165 g. From VAM 60 g was partitioned in to various fractions according to polar escalation: VAH, VAE, VAB and VAA and the yield obtained was 9.891 g, 1.954 g, 9.672 g and 32.163 g, respectively. 3.2. HPLC-DAD analysis of VAE The HPLC-DAD analysis of VAE indicated the presence of rutin (3.593±0.21 µg/mg), gallic acid (0.246±0.09 µg/mg), caffeic acid (0.587±0.12 µg/mg), apigenin (0.527±0.11 µg/mg), myricetin (1.33±0.35 µg/mg) and quercetin (1.23±0.21 µg/mg) as shown in Table 1. The

absorption spectra of VAE are shown in Figure 1. the reproducibility of results was also determined. 3.3. Reproducibility of HPLC results For the reproducibility of HPLC results the samples were run under the same condition for estimation of rutin on five different days. The results indicated RSD% for peak area of 1.59 and for the respective yield of 1.34 (Table S1). 3.4. Acute toxicity studies Results of this study showed that VAM and its fractions did not induce any variation on urination, purgation, diarrhea, food intake, piloerection, salivation, lacrimation, nasal secretion, physical activity, mood and aggression, balance, lesions, signs of toxicity or death in period of two weeks. 3.5. Protection of protein denaturation Results showed that extract/fractions of V. arnottianum protected the protein from heat induced denaturation and notable percent inhibition was shown by VAE (73.000±4.3%) followed by VAM (70.750±4.4%) in comparison to the standards diclofenac potassium (76.000±4.7%) and aspirin (79.48±4.8%) at 500 µg/ml of concentration (Table 2). 3.6. Anti-inflammatory studies in rat 3.6.1. Protection against carrageenan in rat The anti-inflammatory results of VAM and its derived fractions are presented in Table 3. VAM and VAE inhibited the development of edema in a time and dose dependent manner. Administration of VAM and VAE at 300 mg/kg after 1 h of carrageenan injection produced the similar anti-inflammatory effects to that of the standard drug diclofenac potassium used in this study. However, after 2 h, 3 h and 4 h the percent inhibition of edema by the standard drug significantly (p > 0.05) higher as compared to the extract/fractions. The 300 mg/kg dose of VAM and VAE after 4 h of administration strongly inhibited the development of edema (71.261±4.8% and 78.067±4.6%, respectively). On the basis of early screening of antiinflammatory activity VAM and VAE were selected for further evaluation of the antiinflammatory studies. 3.6.2. Effect of V. arnottianum on formalin induced edema Results of the extract/fractions of V. arnottianum for anti-inflammatory activity after formalin induced paw edema are presented in Table 4. The paw volume increased progressively during the period from 1 h to 24 h with formalin in rat. The results indicated that VAM and VAE treatments progressively inhibited the edema induced with formalin up to 6 h and then a decline in inhibition was recorded with all the treatments. The results showed that VAE at

300 mg/kg concentration had produced significant (p < 0.05) anti-inflammatory activity (69.10±0.39%) after 6 h and was followed by VAM (57.31±0.31%) in comparison to Ibuprofen (79.26±0.44%). 3.6.3. Inhibition of croton oil induced ear edema by V. arnottianum Results showed that application of VAM and VAE dose dependently inhibited the gain in weight of ear lobe induced by croton oil in rat (Table 5). The inhibition in gain of weight of ear at the highest dose (5 mg/kg) of VAM and VAE was 64.38±1.8% and 73.12±1.9%, respectively. 3.6.4. Complete Freund's adjuvant-induced inflammation in rats 3.6.4.1. Effect of V. arnottianum on paw volume in treatment mode The inhibitory effects of V. arnottianum on paw volume in treatment mode are presented in Figure 2a. Administration of VAM and VAE to FCA treated rats dose dependently inhibited the development of edema in rat. The effects of VAM and VAE were more pronounced at 300 mg/kg to rat from the 15th to 25th day and displayed significant (p < 0.05) reduction in paw edema. 3.6.4.2. Effect of V. arnottianum on paw volume in preventive mode In preventive mode of treatment VAM and VAE at 150 mg/kg and 300 mg/kg and ibuprofen (10 mg/kg) were given from day 1st after injection of FCA to rat. VAM and VAE at 300 mg/kg dose exhibited similar preventive effects for percent inhibition of edema in FCA treated rats (Fig. 2b). After the injecting of FCA to rat inflammation symptoms develop gradually and peaked between days 11 to 15 (Fig 2c). The inflammation scores were significantly (p < 0.05) decreased in ibuprofen (10 mg/kg) and at 300 mg/kg of VAE and VAM treated group. 3.6.4.3. Protective effects on body weight and organ weight In FCA treated rats significantly lower body weights was recorded as compared to control rats (Fig. 3A). However, FCA co-treated rats with V. arnottianum showed 32.6±3.06 g increase in body weight at 300 mg/kg of VAE (300 mg/kg) and VAM (25.66±1.17 g) against the FCA treated rats. Comparatively lower increase in body weight of rats over the FCA treated rats at 150 mg/kg was recorded by VAM and VAE treatment to rat (Fig. 3-A). FCA treatment to rat significantly (p < 0.05) increased the liver index and decreased the thymus and spleen index whereas nonsignificant alteration in kidney index was recorded as compared with control rats (Fig. 3B). In FCA treated rats co-administration of VAM (300 mg/kg) restored the liver (8.33%), thymus (18.09%) and spleen (11.48%) and VAE (300 mg/kg) restored (13.76%, 26.40%, 17.64%, respectively) as compared to FCA treated rats.

The ibuprofen co-treated rats showed 10.3%, 17.7% and 21.68% recovery for liver, thymus and spleen, respectively. 3.6.4.4. Enzymatic and biochemical regulation by V. arnottianum In FCA treated rats the level of AST, ALT, ALP, urea, creatinine, total bilirubin significantly (p < 0.05) increased while albumin in serum decreased as compared to control group. However, treatment with V. arnottianum extract/fractions significantly (p < 0.05) restored the level of altered enzymes, urea, creatinine, total bilirubin and albumin in serum of rat (Table 6). 3.6.4.5. V. arnottianum effect on FCA-induced endogenous antioxidant enzymes FCA treatment to rat caused significant (p < 0.05) decrease in the level of CAT, POD and SOD while increase in ROS in serum of rat (Fig. 4). The co-administration of VAM and VAE to FCA treated rats reversed these effects and elevated the activity of these enzymes. The level of ROS decreased with co-administration of VAM and VAE to FCA treated rats. Lower concentration of VAM and VAE (150 mg/kg) moderately restored the level of these parameters whereas significant restoration at higher dose was recorded at 300 mg/kg of VAM and VAE to FCA treated rats (Fig. 4). 3.6.4.6. Studies on interleukins and nitric oxide in serum In this study treatment of FCA to rat significantly (p < 0.05) increased the levels of IL-17 (110.93±5.03 pg/ml), IL-6 (96.43±2.05 pg/ml), TNF-α (124.12±8.59 pg/ml) and NO (83.61±3.19 µM/ml) in serum as compared to control rat. Co-treatment of extract/fractions decreased the concentration of above parameters (Fig. 5). 3.6.4.7. Histological studies FCA treatment to rat caused severe edema, abnormal joint architecture, cells infiltration, cartilage destruction and decrease in joint space. However co-administration of VAM and VAE at both 150 mg/kg and 300 mg/kg doses to FCA treated rats showed protective effects on altered histology and joint architecture (Fig. 6). 4. Discussion Medicinal plants contain antioxidants which play important role to scavenge the free radicals. Six polyphenolics rutin, gallic acid, caffeic acid, apigenin, myricetin and quercetin were found in the extract. Rutin was present in maximum quantity followed by myricetin and quercetin. Rutin showed admirable antioxidant, anti-inflammatory and hepatoprotective activities (Kubola and Siriamornpun, 2011). Naturally occurring plant phenol, gallic acid, also exhibits strong antioxidant potential during in vitro assays (Khan et al., 2015; Yen et al., 2002). Caffeic acid has been identified as one of the active antioxidant (Dalgarno et al.,

2005). Quercetin and myricetin are the other antioxidants found in VAE (de Whalley et al., 1990). The antioxidant potential of VAE might be attributed by the presence of bioactive polyphenolic and other antioxidant constituents. Presence of antioxidant compounds including quercetin, apigenin and caffeic acid has also been reported in Vincetoxicum lutea (Šliumpaitė et al., 2013). This study also reported in vitro antioxidant activity of V. lutea extracts. Inflammation is attributed as vasodilatation, vascular permeability, increased blood flow and cellular recruitment to the site of inflammation. Prostanoids such as prostaglandins and thromboxanes are quickly produced via the cyclooxygenase pathway in different body cells in response to numerous stimuli including inflammation (Hata and Breyer, 2004). Prostaglandins, nitric oxide (NO) along with cytokines such as TNF-α and 1L-6 are the key inflammatory mediators. Cyclooxygenase (COX) is present in at least two key isoforms, COX-1 and the other inducible COX-2. COX enzymes catalyse prostaglandins and thromboxanes synthesis from arachidonic acid that is released by many cell types (Jin et al., 2010). In inflammatory lesions, the inducible nitric oxide synthase (iNOS) is a key player in producing large amounts of NO. During acute inflammation synthesis of endothelial nitric oxide synthase (eNOS) enhances the vascular leakage (Bucci et al., 2005). Therapeutic potential of various agents against inflammation has been evaluated in different studies (Afsar et al., 2015; Jan and Khan, 2016; Maryam et al., 2019). Carrageenan induced inflammation model demonstrated two phases for edema development. During early phase (0−1 h) vascular permeability occur by release of histamine, 5-hydroxytryptamine and bradykinin, while in 2nd phase (1– 6 h) enhanced level of prostaglandins and inducible cyclooxygenase (COX-2) were recorded (Di Rosa et al., 1971). The conditions become more severe by infiltration of neutrophils and consequently the generation of ROS in the inflamed area (Moncada and Higgs, 1993). The present study indicated a marked anti-inflammatory effect of VAE during both initial and late phase of edema development possibly by inhibiting the release of specific mediators. These anti-inflammatory effects of VAE might be attributed by the presence of different polyphenolic and other bioactive constituents which inhibited the infiltration of neutrophils, and suppressed the release of NO, TNF-α and IL-1β. Decrease in edema has been studied with mixture of plants extract containing Vincetoxicum officinalis (Penkov et al., 2014). Production of inflammatory mediators was suppressed by Cynanchum wilfordii (Cho et al., 2017). Application of C. atratum in atopic dermatitis model inhibits the synthesis of

inflammatory cytokines (Choi et al., 2017). Anti-inflammatory potential of extract/fractions was also reported in earlier studies (Sajid et al., 2017; Maryam et al., 2019). Formalin induced edema is also a biphasic event; initially there is the release of bradykinin and substance P whereas secretion of serotonin, histamine, bradykinin, and prostaglandins occur in the late phase (Sreejamole et al., 2011). In this study administration of VAM and VAE (150 mg/kg; 300 mg/kg) attenuated the development of paw edema and these effects are lasted for 24 h. These results suggest the therapeutic potential of extract/fraction in formalin induced edema development. Anti-inflammatory effect of V. arnottianum extract/fractions on the formaldehyde test, particularly in the neurogenic phase show the promising result as compared to standard drug used. Croton oil is constituted of 12-o-tetracanoilphorbol-13-acetate (TPA) and several other phorbol esters as chief irritants. Use of croton oil on ear causes induction of protein kinase C which result in activation of other enzymatic pathways including mitogen activated protein kinases, phospholipase A2 and release of arachidonic acid and platelet activation factor. This mechanism contribute towards vasodilation, polymorphonuclear leukocytes migration, vascular permeability, release of serotonin, histamine and restrained synthesis of inflammatory eicosanoids by 5-5- lipoxygenase (LOX) and COX enzymes (Wang et al., 2001). During allergic reactions of various organs including skin, nose, throat and lungs release of histamine occur that regulate the migration and function of lymphocytes and neutrophils (Zampeli and Tiligada, 2009). Application of VAM and VAE at 2.5 mg/ear and 5 mg/ear markedly inhibited the development of ear edema induced with croton oil in rat. The inhibition of ear edema with extract/fractions of Wendlandia heynei was reported earlier (Maryam et al., 2019). At the FCA inflamed area infiltration of macrophages and polymorphonuclear cells occur resulting in synovitis and generation of ROS. This has resulted in activation of inflammatory molecules which severely adverse the situation that leads to progression of inflammatory scores. FCA induced inflammation simulates the characteristics of human rheumatoid arthritis patients related to immunology and histology (Simoes et al., 2005; Jawed et al., 2010). In this animal model activation and release of pro-inflammatory cytokines and interleukins altered the osteoclast differentiation and consequently result in bone erosion (Sajid et al., 2017). In this investigation higher level of TNF-α, IL-1β, IL-17 and IL-6 was recorded in the FCA treated rats. These cytokines regulate the stimulation of mesenchymal cells like osteoclast, synovial fibroblast and chondrocytes. The rats co-treated with VAM and VAE decrease the level of these pro-inflammatory cytokines in the preventive mode

throughout the study period. The anti-inflammatory activity might be attributed by the higher concentration of rutin, gallic acid, quercetin and phytol in the extract. The antioxidants with free radical scavenging activities found in plants also showed promising aptitude to inhibit the activity of inflammatory enzymes including lipoxygenase, prostaglandin synthase and cyclooxygenase (Miean and Mohamed, 2001). These chemicals might inhibit the infiltration of neutrophils and consequently the decrease in TNF-α and IL-1β levels and oxidative stress (Silva et al., 2014). Similar anti-inflammatory results of plant extract for suppression of proinflammatory cytokines were reported in earlier studies (Younis et al., 2016; Maryam et al., 2019). Another important parameter to determine the FCA induced inflammation was the decrease in body weight and indices of thymus, kidneys and spleen. However, the index of liver was increased with FCA treatment to rat. These results showed that altered body weight and indices of various organs might be due to progression of inflammatory cytokines. The coadministration of FCA treated rats with VAM and VAE restored the altered parameters towards the control rats. These results suggest an alternative therapeutic agent for inflammation related disorders. Oxidative stress was caused by FCA which generates free radicals and cause tissue injury by DNA damage, disturbed enzymatic level and elevated lipid peroxidation. In the current study arthritic rats showed elevated levels of liver serum markers; ALT, AST, ALP and biochemical parameters like albumin and bilirubin due to damaged hepatocellular membrane integrity. Ibuprofen treated group displayed the great cure as the levels of liver marker closed to normal values. The VAM and VAE showed its protective capacity per its dose concentration. The high dose (300 mg/kg) was more protective. Liver serum levels of AST, ALT, ALP is associated with membrane damage of liver cells, because of cytoplasmic location of above enzymes that are leaked out into plasma due to damaged membrane. Nature has equipped the individuals to counter balance the free radicals; antioxidant enzymes, reduced glutathione and many other molecules. If this defense unable to eradicate the ROS then it necessitated the use of external compounds for protection against free radical induced damages. In the present study, FCA injection to rat causes oxidative stress and decrease level of CAT, POD and SOD was recorded (Majid et al., 2018). The protective effects of VAM and VAE restored the level of antioxidant enzymes such as CAT, SOD and POD that was declined due to FCA induced inflammation in rat. The radical scavenging activities of the polyphenols present in VAM and VAE might ameliorate the FCA induced stress in rat and consequently inhibit the edema development on rat.

5. Conclusion The phytochemical, in vitro and in vivo antioxidant and anti-inflammatory studies of the extract/fractions of V. arnottianum were performed in the present work. The results demonstrate that the chemical composition and biological activities of the extract were dependent on the polarity of the solvent. Rutin was found as the major phenolic component in the ethyl acetate fraction. All the extract/fractions have shown in vitro anti-inflammatory activity. However, the ethyl acetate fraction deliberated the significant anti-inflammatory and antioxidant potential as compared to the methanol extract; suggested a new therapeutic agent for inflammation related disorders. Acknowledgements MRK is intensely acknowledged for his kind supervision, expert guidance and substantial facilitations of all necessary materials and equipment. Funding The project was funded by the Department of Biochemistry Quaid-i-Azam University Islamabad Pakistan. Author contributions ZZ, SAS, SM, MM, TY and MS carried out experimentation. ZZ and MRK executed the data analysis and write the manuscript. Competing interest The authors declare that they have no competing interests.

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Figure 1: HPLC-DAD profile of V. arnottianum ethyl acetate fraction at different wavelengths. Signal 1: 257λ, Signal 2:279λ, Signal 3: 325λ, Signal 4; 368λ.Conditions: Injection volume 20 µl, Flow rate 1 ml/min, Agilent RP-C8. Figure 2: Assessment of FCA induced edema and scores. Data is presented as mean ± SD (n= 3). (a): Treatment mode of studies where the induction was done till day 13 and then dose was started. Edema in arthritic control group was increasing with passage of time. VAM and VAE 300 mg/kg significantly decreased arthritic edema after 15th day. (b): Preventive mode of studies where dose was given after inducer at day 1. Arthritic control group has not prevented the induction of the disease while VAE and VAM at 300 mg/kg have significantly prevented arthritic edema. (c): Arthritic score where arthritic control group has higher arthritic score compared to normal and other groups. The ibuprofen treated animals showed significant decrease in arthritic score after first week to onward in intended time of experiment. Figure 3: Assessment of change in body weight and organ indices in experimental animals. (A): Variation in mean body weight over time, (B): Normal and FCA treated organ indices. Each value represents the mean ± SD (n=3). Differences were considered significant at the level of p < 0.05. Figure 4: Effect of V. arnottianum fractions; VAM and VAE at 150 mg/kg and 300 mg/kg on FCA stimulated endogenous antioxidant enzymes in serum of experimental animals. Catalase (CAT), Superoxide dismutase (SOD), Peroxidase (POD) and the level of Reactive oxygen species (ROS). Data is presented as mean±SD (n = 3). Figure 5. Effect of V. arnottianum ractionson FCA stimulated IL-1β, IL-6 IL-17, TNF-α and NO levels in experimental animals. Data is presented as mean±SD (n = 3). Differences were considered significant at p < 0.01. (I); Normal control, (II); FCA treated rat, (III); Ibuprofen, (IV); VAM (150 mg/kg) (V); VAM (300 mg/kg) (VI); VAE (150 mg/kg), (VIX); VAE (300

mg/kg). VAM; V. arnottianum methanol extract, VAC; V. arnottianum chloroform fraction, VAE; V. arnottianum Ethyl acetate fraction. Figure 6: Investigation of histological architecture of paw edema induced with FCA. (A): normal rat, (B): Ibuprofen 10 mg/kg, (C): FCA treated rat, (D): VAM 300 mg/kg, (E): VAM 150 mg/kg, (F): VAE 300 mg/kg, (G): VAE 150 mg/kg. The black double arrow showing variation in joint spaces among different groups, while single arrows represent the chondrocytes or cartilage erosion.

Supplementary file Table S1

Table 1: HPLC-DAD results for ethyl acetate fraction of V. arnottianum Constituent

Signal wavelength (nm)

Quantity (µg/mg dry extract)

Rutin

257

3.593±0.21

Gallic acid

279

0.246±0.09

Caffeic acid

325

0.587±0.12

Apigenin

325

0.527±0.11

Myricetin

368

1.331±0.35

Quercetin

368

1.231±0.21

Each value is represented as mean±SD (n=3).

Table 2: In vitro inhibition of heat induced denaturation of albumin of V. arnottianum Percent inhibition of protein denaturation at various concentrations (µg/ml) Sample

100

200

300

400

500

VAM

48.500±1.3ab

55.250±2.5ab

61.000±3.1ab

67.750±3.9b

70.750±4.4ab

VAH

42.750±1.4bc

47.750±1.9bc

55.250±2.9bc

58.500±3.1c

62.250±3.8bc

VAE

50.250±1.7ab

59.250±2.4a

65.000±3.4a

68.750±3.9ab

73.000±4.3ab

VAB

39.750±1.1c

42.750±1.8c

47.750±2.8cd

52.250±3.1d

55.250±3.8c

VAA

27.500±1.2d

37.500±1.4c

44.750±2.3d

47.750±2.9d

52.750±3.2c

Aspirin

52.500±1.5a

60.833±2.4a

67.000±3.6a

74.000±4.1a

79.917±4.8a

DICP

52.417±1.8a

60.083±2.5a

64.167±3.5a

73.083±4.0ab

76.000±4.7a

Effect of V. arnottianum on heat induced albumin denaturation. VAM; V. arnottianum methanol extract, VAH; V. arnottianum n-hexane fraction of VAM, VAE; V. arnottianum ethyl acetate fraction of VAM, VAB; V. arnottianum butanol fraction of VAM, VAA; V. arnottianum residual aqueous fraction of VAM, DICP; diclofenac potassium. Each value is represented as mean ± SD (n = 3). Means with different superscript (a-d) letters in the column are significantly (p < 0.05) different from one another.

Table 3: Effect of V. arnottianum on carrageenan induced paw edema in rat Treatment

Dose

Mean paw volume before carrageenan injection

Change in edema volume (ml) after carrageenan injection (mean ± SD)/ Percent inhibition of edema + 1h

+2h

+3 h

+4 h

Saline

1 ml/kg

1.15±0.1

1.37±0.27

1.26±0.48

1.20±0.37

1.00±0.28

10% DMSO

1 ml/kg

1.06±0.07

1.33±0.09 (13.82±2.1i)

1.08±0.07 (24.17±2.2j)

0.90±0.03 (32.93±.2k)

0.67±0.02 (40.88±3.8j)

VAM

150 mg/kg,

1.05±0.06

0.85±0.07 (26.97±2.6cd)

0.63±0.05 (42.67±2.4gh)

0.49±0.04 (50.89±3.5hi)

0.31±0.03 (64.02±4.1h)

300 mg/kg,

1.09±0.04

1.09±0.1 (29.450±2.3abc)

0.70±0.07 (50.949±2.9cde)

0.51±0.04 (61.646±3.8de)

0.35±0.03 (71.261±4.8d)

150 mg/kg,

0.86±0.03

0.94±0.08 (19.061±1.6hi)

0.74±0.06 (32.99±2.1j)

0.58±0.04 (47.051±3.1j)

0.39±0.03 (58.434±3.7i)

300 mg/kg

1.11±0.08

1.15±0.11 (25.47±1.8de)

0.71±0.06 (49.751±3.1de)

0.52±0.04 (61.616±3.6de)

0.32±0.01 (69.622±4.6de)

150 mg/kg

0.86±0.08

0.85±0.08 (27.03±2.3bcd)

0.51±0.06 (53.11±3.1bcd)

0.48±0.04 (63.235±3.9c)

0.28±0.02 (74.189±4.6c)

300 mg/kg

0.89±0.05

1.09±0.1 (29.172±2.7abc)

0.64±0.07 (55.055±3.3b)

0.50±0.51 (64.086±4.1b)

0.24±0.041 (78.067±4.6b)

150 mg/kg

0.97±0.08

0.90±0.09 (22.02±2.4fgh)

0.66±0.7 (39.48±2.5hi)

0.60±0.05 (47.065±3.8fg)

0.48±0.03 (60.021±4.1efg)

300 mg/kg

1.03±0.09

1.14±0.11 (25.932±2.4d)

0.75±0.8 (47.552±2.7ef)

0.55±0.5 (58.785±3.7ef)

0.39±0.4 (65.983±4.2ef)

150 mg/kg

0.93±0.06

0.90±0.08 (22.66±2.5efg)

0.69±0.06 (36.494±2.9i)

0.61±0.06 (46.203±3.5ij)

0.47±0.033 (58.198±4.2gh)

300 mg/kg

1.07±0.05

1.16±0.02 (24.82±2.3def)

0.87±0.071 (38.561±3i)

0.58±0.05 (53.209±3.6hi)

0.49±0.04 (56.908±3.8i)

10 mg/kg,i.p

1.10±0.09

1.07±0.1 (30.374±2.6a)

0.57±0.06 (60.140±3.2a)

0.37±0.04 (72.029±4.8a)

0.16±0.01 (85.630±5.2a)

VAH

VAE

VAB

VAA

DICP

Effect of V. arnottianum on carrageenan induced paw edema in rats. VAM; V. arnottianum methanol extract, VAH; V. arnottianum n-hexane fraction of VAM, VAE; V. arnottianum ethyl acetate fraction of VAM, VAB; V. arnottianum butanol fraction of VAM, VAA; V. arnottianum residual aqueous fraction of VAM, DICP; diclofenac potassium. Percent inhibition represented as mean ± SD (n=7). Means with different superscript (a-h) letters in the column are significantly (p < 0.05) different from one another.

Table 4: Effect of V. arnottianum on formalin-induced paw edema in rat

Edema volume (ml)/Percent inhibition of edema Samples

Dose

+1 h

+3 h

+6 h

+12 h

+24 h

0.48±0.07

0.7±0.06

0.82±0.08

0.86±0.09

0.92±0.07

(150 mg/kg)

0.39±0.03 (18.05±0.11d)

0.51±0.03 (26.19±0.19e)

0.47±0.02 (41.86±0.21e)

0.52±0.02 (38.75±0.23e)

0.59±0.04 (35.50±0.17d)

(300 mg/kg)

0.35±0.02 (26.38±0.12c)

0.44±0.02 (37.14±0.21cd)

0.35±0.01 (57.31±0.31c)

0.38±0.03 (55.03±0.26c)

0.43±0.04 (52.89±0.22b)

(150 mg/kg)

0.35±0.02 (25.69±0.11c)

0.44±0.02 (37.28±0.2cd)

0.38±0.06 (53.25±0.27d)

0.44±0.02 (48.44±0.25d)

0.49±0.02 (46.01±0.29c)

(300 mg/kg)

0.31±0.01 (35.41±0.15b)

0.39±0.04 (43.80±0.24b)

0.25±0.02 (69.10±0.39b)

0.36±0.02 (58.13±0.26b)

0.41±0.02 (54.71±0.34b)

(10 mg/kg)

0.25±0.01 (47.91±0.2a)

0.3±0.01 (57.14±0.27a)

0.17±0.02 (79.26±0.44a)

0.22±0.03 (74.41±0.41a)

0.26±0.04 (71.73±0.4a)

Formalin F+VAM

F+VAE

F+Ibupro fen

Effect of V. arnottianum on formalin induced paw edema in rats. VAM; V. arnottianum methanol extract, VAE; V. arnottianum ethyl acetate fraction of VAM. Each value is represented as mean ± SD (n = 3). Means with different superscript (a-g) letters in the column are significantly (p < 0.05) different from one another.

Table 5: Effect of V. arnottianum on croton oil-induced ear edema in rat Groups

Normal control Croton oil control CO+VAM

Dose/route

Weight of left ear (mg) 80.04±2.3

Weight of right ear (mg) 80.01±2.5

93.50±3.2

99.67±3.6

Edema (∆ mg)

% reduction of inflammation

2.5 mg/ear 85.64±2.1 88.58±2.8 2.91±0.68 52.29±1.5e 5 mg/ear 87.78±2.3 89.98±2.9 2.21±0.66 64.38±1.8c CO+VAE 2.5 mg/ear 80.81±2.3 83.36±2.7 2.55±0.79 58.71±1.3d 5 mg/ear 86.84±2.5 88.50±2.9 1.66±0.88 73.12±1.9b CO+Ibuprofen 0.5 mg/ear 84.70±2.1 85.80±2.6 1.11±0.71 82.19±2.1a Data values shown represent mean ± SD (n = 3). VAM; V. arnottianum methanol extract, VAE; V. arnottianum ethyl acetate fraction of VAM, CO; Croton oill. Means with different superscript (a-d) letters in the column are significantly (p < 0.05) different from one another.

Table 6: Enzymatic and biochemical investigation of control and FCA treated rats Groups

Dose/route ALT (U/l)

Normal control FCA VAM VAE Ibuprofen

150 mg/kg

AST (U/l)

ALP (U/l)

Urea (mg/dl)

39.00±0.50e

26.7±0.64f 119.00±2.3.0h 32.51±1.89f

93.37±2.40a

97.90±2.6a 334.00±7.01a

Creatinine Total (mg/dl) Bilirubin (mg/dl) 0.44±0.06h 0.41±0.23a

Albumin (mg/dl) 5.81±0.3a

77.34±2.34a 2.04±0.22a

1.92±0.21g

1.99±0.1i

73.16±2.92b 63.23±2.6b 188.38±5.80b 68.71±2.30b 0.93±0.11b

0.83±0.07f

3.55±0.2h

e

4.12±0.4e

0.88±0.09e

0.78±0.09d

4.03±0.4f

d

53.34±2.4

c

159.48±3.87

d

59.12±2.87 60.30±2.8c

c

0.86±0.07

d

300 mg/kg

58.21±2.70

0.76±0.08

150 mg/kg

55.34±2.3de

52.34±2.3c 152.33±5.02e

300 mg/kg

47.44±2.1fg

43.40±2.1d 135.44±4.38g 48.44±2.1de

0.73±0.08g

0.69±0.07c

4.56±0.3c

10 mg/kg

43.44±2.10e

36.92±1.7e 123.34±2.87h 43.08±1.9e

0.66±0.07g

0.59±0.08b

4.98±0.4b

VAM; V. arnottianum methanol extract, VAE; V. arnottianum ethyl acetate fraction of VAM, FCA; Freund’s complete adjuvant. Data values shown represent mean ± SD (n = 3). Means with different superscript (a-g) letters in the column are significantly (p < 0.05) different from one another.