Anti-inflammatory potential of dichloromethane leaf extracts of Eucalyptus globulus (Labill) and Senna didymobotrya (Fresenius) in mice

Background Inflammation is an immune response characterized by swelling, redness, pain and heat. Inflammation is mainly managed using conventional medicines that are associated with many side effects. Plant-based remedies are considerably better alternative therapies for they have fewer side effects. Objective This study aimed at determining the anti-inflammatory potential of dichloromethane (DCM) leaf extracts of Eucalyptus globulus and Senna didymobotrya in mice. Methods Fresh leaves of these plants were harvested from Embu County, Kenya. Quantitative phytochemical analysis was done using Gas Chromatography-Mass Spectrometry (GC-MS). Anti-inflammatory test comprised nine groups of five animals each: normal, negative, positive controls and 6 experimental groups. Inflammation was induced with Carrageenan. One hour post-treatment, the different groups were intraperitoneally administered with the reference drug, diclofenac, 3% DMSO and six DCM leaf extracts at doses of 25, 50, 100, 150, 200 and 250mg/kgbw. Results GC-MS results revealed α-phellandrene, camphene, terpinolene, and limonene among others. Anti-inflammatory effects showed that extract doses of 100,150,200 and 250mg/kg bw significantly reduced the inflamed paw. Doses of 200 and 250mg/kgbw in both plants were more potent and compared with diclofenac. E. globulus extract dose of 250mg kg bw reduced inflamed paw in the 1st, 2nd, 3rd and 4th hours, by 2.27,6.52,9.09 and 10.90% respectively while S.didymobotrya at similar dose ranges, inflamed paw reduced by 2.41, 5.43, 8.31 and 9.05% respectively. Conclusion E. globulus and S. didymobotrya have potent anti-inflammatory activities, attributed to their constituent phytochemicals. This study confirms the traditional use of these plants in treating inflammation.


Introduction
Inflammation is a complex biological reaction of the vascular tissues to noxious stimuli like damaged cells, irritants and pathogens 1 . The objective of the inflammatory response is to limit and remove the causative agents of inflammation as well as to initiate the healing process in the damaged cells 2 . Inflammation is mainly managed using NSAIDs such as Ibuprofen, Naproxen Sodium and Diclofenac among others 3 . These drugs are associated with numerous side effects in the gastrointestinal tract, kidney, liver, cardiovascular system among others 4 . Inflammation is also managed using medicinal plants such as Azima tetracantha (Lam) 5 , Eugenia jambolana 6 among others. Herbal medicines serve as better al-ternatives for they are arguably readily available, cheap and possess fewer side effects hence the need to search for new bioactive compounds in medicinal plants 7 . E. globulus is an evergreen tree with a straight trunk of about 0.6 to 2 meters in diameter and a height of 40-70 meters tall. It has well spread deep roots along with smooth, brownish, greenish, mottled gray and long peeling bark. Most of them grow naturally in many parts of Kenya 8 . E. globulus is used in the management of abscess, asthma, burns, sore throat, bronchitis, colds, among others 9 . S. didymobotrya was introduced as an ornamental plant and grow in almost every part of Kenya. It is a bushy shrub found at altitudes of between 1450-2400 meters above the sea level. The plant grows to a maximum height of about 30-90cm 10 . It is used in the management of fungal, bacterial, parasitic infections, hypertension among others 11 . In Embu County, Kenya, E. globulus and S. didymobotrya are used in the traditional medicine in treatment of inflammation. However, there is no scientific data to confirm and/or validate this use. This study sought to explore the anti-inflammatory potential of dichloromethane leaf extracts of E. globulus (Labill) and S. didymobotrya (Fresenius) in mice models.

Materials and methods Plant samples harvesting, processing, and extraction.
The researchers obtained research authorization to undertake this study from National Commission for Science, Technology and Innovation, Kenya (NA-COSTI/P/16/6765/1452). The collection of the plant samples was carried out based on ethnobotanical information availed by a local herbalist in Mbeere North Subcounty, Embu County,Kenya. Fresh leaves of E. globulus (Labill) and S. didymobotrya (Fresenius) were collected at GPS location of 0o35'12."S, 37o38'32"E and 0o35'28."S, 37o38'25"E respectively. The samples were cleaned, enveloped in Khaki bags and carried to Kenyatta University in the Department of Biochemistry, Microbiology and Biotechnology, where the study was undertaken. The samples were identified by Mr.
Stephen Mwangi, a taxonomist in Kenyatta University. Voucher specimens of E. globulus (Labill) and S. didymobotrya (Fresenius) were deposited at the Plant Sciences Herbarium of Kenyatta University and were assigned voucher specimen numbers JKM001 and JKM002, respectively.
The samples were air-dried at 25ºC in the plant drying facility of Kenyatta University for 14 days. The dried samples were then milled separately into fine powders using an electric mill. The powders were stored at room temperature in well-sealed and labeled khaki papers awaiting extraction 12 .
For extraction, six hundred grams of each plant powder were separately weighed and put into well-labeled conical flasks. Two liters of dichloromethane were added into each conical flask, corked and left to stand for 24 hours with regular swirling every 6 hours. The mixtures were then decanted in clean and well labeled conical flasks. Filtration was then done using Whatman No.1 filter papers. Each filtrate was put in a well-labeled conical flask. One liter of dichloromethane was added to each residue and then left to stand for 24 hours, followed by decantation and filtration. This procedure was repeated until dichloromethane remained clear. Each extract was concentrated using a rotary evaporator at 40ºC until dry. The semi-solid extract was separately put in an open clean beaker for 5 days to evaporate the remaining solvent. A sticky solid was obtained and store stored at -4°C until use 13 .

GC-MS analysis
Quantitative phytochemical analysis of the extract was done at the International Centre of Insect Physiology and Ecology (ICIPE) laboratories. A protocol reviewed by the Principal Scientist and Head of the Department of Behavioral and Chemical Ecology at ICIPE, Prof. Baldwyn Torto, was used to analyze the two plant extracts. The DCM extracts of E. globulus (Labill) and S. didymobotrya (Fresenius) DCM weighing 1.1 and 1.2mg respectively were diluted in their respective volumes through partitionng between hexane and methanol. This was followed by vortexes and centrifugation. Bypassing through anhydrous Na 2 SO 4 , the hexane layer was dried and then analyzed using GC-MS. Standard authentic serial dilutions (1,8-cineole; 99%,Gillingham,-Dorset, England) (50ng/µl, 150ng/µl, 250ng/µl, 350 ng/µl and 550 ng/µl) were prepared, analyzed using GC-MS and their peaks used in quantification. The calibration curve of 1,8-cineole (peak area vs. concentration), with the equation y=7E+06-1E+07, served as the basis for the external quantification of the target compounds Figure 1.
Agilent Gas Chromatograph 7890A/5975C coupled to Mass Spectrometer in full scan mode was used to analyze the two plant extracts using the following specifications; gas chromatography Column (HP-5 MS low bleed capillary column (0.25μm, 30 m by 0.25 mm i.d.) (J and W, Folsom, California, United States of America), flow rate, (Helium) (constant flow mode at 1.25 ml/ min), injection split mode, the temperature of the oven (35°C) for initial 5 minutes and then raised by 10°C per minute to 280°C for 10.5 minutes; run time 70 minutes 14 . The percentage abundance of each phytochemical identified by GC-MS was computed using the formula below;

Experimental animals
Swiss albino mice weighing between 20 to 25 grams and aged 5-6 weeks were used in the present study. They were obtained from Kenyatta University Animal Breeding Facility. The mice were provided with laboratory diet, which comprised rodent pellets and water ad libitum. The mice were housed in groups of nine in standard polypropylene cages and maintained at room temperature (23 ± 2°C), bench level lighting of 360 lux and humidity (55 ± 5%) with 12hrs light and 12hrs dark cycle. Approval for use of laboratory animals in this study was obtained from the Ethics Committee for the Care and Use of Laboratory Animals of Kenyatta University, Kenya (KU/AERC/107-012/2017). Development of the experimental protocols and procedures were performed under the guidance of the Veterinarian, who is a member of the Kenya Veterinary Board (KVB). All procedures were carried out following the Public Health Service (PHS) Policy on Humane Care and Use Committee (IACUC) (Section 8.3.2) and KVB.

Induction of inflammation
To induce inflammation, each mouse was injected with 0.1ml of 1% Carrageenan (commercial grade type 1) solution in normal saline in the left hind paw. Induction of inflammation was indicated by the manifestation of edema on the hind paw 15 .

Experimental design
A completely randomized controlled study design was adopted in this study from which an experimental design was derived. Swiss albino mice were categorized into 9 sets of 5 mice each. The animals received treatments as follows; Group I (normal control) mice received 3% DMSO only and were not induced with inflammation 16 . Group II (negative control) mice were induced with inflammation and administered with 3% DMSO after one hour 17 . Group III (positive control) animals were induced with inflammation and then treated with the reference drug diclofenac sodium at a dose of 15mg/kg body weight after one hour 18 . Animals in groups (IV, V, VI, VII, VIII, and IX) were induced with inflammation and then treated with extracts dosages of 25, 50, 100, 150, 200 and 250mg/kg bw respectively after one hour. All the treatment doses were freshly prepared. The summary of this design is detailed in Table  1. The paw circumference (edema) was determined one hour post induction of inflammation and at hourly in- tervals after administration of different treatments for four hours. The paw circumference was measured using cotton thread and then transferred to the ruler to obtain the reading in millimeters 19 . The percentage edema inhibition was computed using the formula described by Umamageswari and Kudagi (2015) 20 .
Where, Vc is the Mean edema volume in the control group and Vt is the Mean edema volume in the treated group.

Statistical data analysis
Raw data on inflammation was obtained, recorded and entered into a spreadsheet. Descriptive statistics were computed and the data expressed as mean± SEM. Inferential statistics were done using One-Way Analysis of Variance (ANOVA), followed by Tukey's post hoc test for pairwise comparison and separation of means. The anti-inflammatory activities of the two DCM plant extracts wre compared using the unpaired student t-test. The level of significance was set at 99.5% (p≤0.005).

Quantitative phytochemical analysis of anti-inflammatory compounds of E. globulus and S. didymobotrya.
The GC-MS analysis of the DCM leaf extract of E. globulus (Labill) revealed the presence of several bioactive chemical compounds that have potential anti-inflammatory activities ( Table 2).  (Table 3). However, the extract dose of 25mg/kg bw did not reduce inflammation (Table 3). At this hour, the anti-inflammatory activity of E. globulus extract at doses of 50, 100, 150, 200 and 250 mg/kg bw was statistically similar and comparable to the activity of diclofenac sodium as presented in Table 3 (p > 0.005).
In the second hour, the E. globulus (Labill) extract, at the doses of 50, 100, 150, 200 and 250 mg/kg bw, reduced the paw edema in mice by 1.87%, 3.86%, 4.70%, 5.90% and 6.52% respectively (Table 3). However, at this hour, the E. globulus (Labill) extract dose of 25 mg/kg body weight exhibited no anti-inflammatory effects. There was a significant variation in the anti-inflammatory effect of E. globulus extract at the six dosages (p < 0.005; Table 3). However, the anti-inflammatory activity of diclofenac was not significantly different compared to that of DCM extract of E. globulus (Labill) at the dose levels of 200 and 250 mg/kg body weight as presented in  Table 3). Generally, the E. globulus extract exhibited a dose-dependent response at all the hours tested (Table 3). Notably, the percentage paw circumference in the negative control group mice was significantly higher compared to those of extract-treated mice as well as those of rats in the positive control and normal control groups (p < 0.005; Table 3). On the other hand, the DCM leaf extract of S. didymobotrya, at the six tested doses demonstrated anti-inflammatory effects on carrageenan-induced inflammation in Swiss albino mice (Table 4). In the first hour, the DCM extract of S. didymobotrya extract doses of 100, 150, 200 and 250 mg/kg bw decreased the inflamed paw circumference in mice by 1.11%, 1.40%, 2.14% and 2.41% respectively. However, the extract never exhibited anti-inflammatory effects at 25 and 50mg/kg body weight dose levels ( Table 4). The anti-inflammatory activities of the S. didymobotrya extract doses of 100, 150, 200 and 250 mg/kg body weight exhibited no significant differences (p > 0.005; Table 4). Similarly, the anti-inflammatory activity of the reference drug (diclofenac) was comparable to those of S. didymobotrya extract doses of 150, 200 and 250mg/kg bw (Table 4; p > 0.005). In the 2 nd hour, the DCM leaf extract of S. didymobotrya, at doses of 50, 100, 150, 200 and 250 mg/kg bw, decreased the inflamed paw circumference in mice by 0.50%, 2.54%, 3.71%, 4.12% and 5.13% respectively (Table 4). However, the extract dose level of 25mg/kg body weight never revealed anti-inflammatory activity ( Table 4). The anti-inflammatory activity of the tested doses of S. didymobotrya extract exhibited significant differences in mice (p < 0.005; Table 4). However, the anti-inflammatory activity of diclofenac was statistically insignificant compared to that of S. didymobotrya at the doses of 200 and 250 mg/kg bw (p > 0.005; Table 4). In the 3rd hour, the S. didymobotrya extract at doses of 25, 50, 100, 150, 200 and 250 mg/kg bw reduced the inflamed paw circumference by 1.13%, 1.99%, 3.81%, 5.11%, 5.50% and 8.31% respectively ( Table 4). The anti-inflammatory effects of S. didymobotrya extract exhibited significant differences at the six tested doses at this hour (p<0.005; Table 4). However, the effect of the diclofenac was comparable to that of the DCM extract of S. didymobotrya at the dose level of 250mg/kg bw in Swiss albino mice (p > 0.005; Table 4). The S. didymobotrya extract doses of 25, 50, 100, 150, 200 and 250 mg/kg body weight reduced the inflamed paw circumference by 2.59%, 3.16%, 4.61%, 6.34%, 7.03% and 9.05% respectively in the fourth hour (Table  4). At this hour, there was a significant variation in the anti-inflammatory effects of S. didymobotrya extract in mice at the six tested doses (p<0.005; Table 4). However, the anti-inflammatory effect of the reference drug, diclofenac, was comparable to that of extract at dose of 250mg/kg body weight (p >0.005; Table 4). Generally, the percentage paw circumference in the negative control group mice was significantly higher compared to extract-treated mice as well as mice in the positive and normal control groups (p < 0.005; Table 4). Comparison between anti-inflammatory activities of DCM extracts of E. globulus (Labill) and S. didymobotrya (Fresenius) In comparison, the anti-inflammatory activities of the DCM leaf extracts of the two plant extracts in mice, at the dose of 25mg/kg body weight, were not significantly different in the four hours of the test period (p > 0.005; Figure 2). Comparatively, the anti-inflammatory activity of the E. globulus extract dose level of 50mg/kg body weight was significantly higher compared to that of S. didymobotyra in the first, second and third hours at the same dose lev-el (p<0.005; Figure 3). However, at the same dose level, the anti-inflammatory effects of E. globulus (Labill) and S. didymobotyra (Fresenius) extracts showed no significant differences in the fourth hour(p > 0.005; Figure 3). At the dose level of 100mg/kg body weight, the anti-inflammatory effects of DCM leaf extracts of E. globulus and S. didymobotyra were not significantly different in the first hour (p > 0.005; Figure 4). In contrast, the anti-in-flammatory effect of E. globulus extract was significantly higher compared to that of S. didymobotyra at the same dose in the second, third and fourth hours (p≤0.005; Figure 4).

Figure 4
Comparison of anti-inflammatory effects of E. globulus (Labill) and S. didymobotyra (Fresenius) DCM leaf extracts at100mg/kgbw on carrageenan-induced inflammation in mice. Means with different letters are statistically significant in each hour of treatment (p≤ 0.005). bw = bodyweight.
The anti-inflammatory effects of E. globulus and S. didymobotyra extract dose levels of 150mg/kg body weight revealed no significant difference in mice (p > 0.005; Figure 5). However, the anti-inflammatory activity of E. globulus extract at the same dose was significantly higher compared to that of S. didymobotyra in the second, third and fourth hours (p≤0.005; Figure 5). There was insignificant variation in the anti-inflammatory effects of E. globulus and S. didymobotyra extract doses of 200mg/kg body weight in the first and second hours (p > 0.005; Figure 6). However, the anti-inflammatory effects of the E. globlus extract, at the same dose level, were significantly higher than that of S. didymobotyra in the third and fourth hours (p< 0.005; Figure 6). At the dose level of 250mg/kg body weight, the anti-inflammatory effects of DCM leaf extracts of E. globulus and S. didymobotyra in mice were statistically similar in the first, second and third hours (p > 0.005; Figure 7). However, in the fourth hour, there was an insignificant variation in the anti-inflammatory effects of the two plant extracts (p<0.005; Figure 7).

Discussion
The present study was designed to determine anti-inflammatory potential of dichloromethane leaf extracts of E. globulus (Labill) and S. didymobotrya (Fresenius) in mice models. The studied plants are used by Mbeere community in Embu County, Kenya in the management of inflammation but no scientific research data is available to confirm their use.
Inflammation is a complex protective reaction of the body against noxious agents like microorganisms or injured cells 21 . Carrageenan-induced paw inflammation is the most suitable model in the determination of the anti-inflammatory activities of different agents that act by blocking the acute inflammatory mediators in vivo 22 . Carrageenan induces inflammation in two phases 23 . The early phase, which occurs between ninety to one hundred and eighty minutes of the inflammation, is as a result of the discharge of serotonin, histamine among other substances while the late, phase which occurs between 270 to 360 minutes, is postulated to be linked with the activation of the lysosome, prostaglandins and 62 proteases 24 .
The NSAIDs like diclofenac reduce edema by blocking enzyme cyclooxygenase (COX), which catalyzes prostaglandin biosynthesis 25 . Cyclooxygenase enzymes exist in two forms; cyclooxygenase-1 and cyclooxygenase-2. These two enzymes catalyze the synthesis of prostaglandins, which stimulate inflammation. The NSAIDs blocks the cyclooxygenase enzymes and decrease prostaglandin levels in the entire body, leading to a reduction of inflammation 26,27 .
These study findings revealed that the E. globulus (Labill) and S. didymobotyra (Fresenius) DCM leaf extracts possess anti-inflammatory effects, which were evident by the reduction of inflamed paw circumference in mice models after treatment. These findings are consistent with earlier research works on the anti-inflammatory effects of other herbal plants in experimental animals. A study by Owolabi et al. 28 on anti-inflammatory activity of aqueous, ethanol, diethyl ether, n-Hexane root extracts of Feretia apodanthera on carrageenan-induced paw edema in rats, reported that this extract provided great relief by lowering edema in the inflamed paw. A study by Matthew et al. 29 , who carried out studies on pharmacological activities of ethanol and aqueous extracts of dried stem of plant Kalanchoe pinnata (Lam.) Pers against inflammation in rats revealed potent anti-inflammatory activities.
The anti-inflammatory effects of these plants are attributed to their constituent phytochemicals like terpenoids, flavonoids and essential oils. Other researchers have associated these phytochemicals with anti-inflammatory activities. A review by Andrade and De Sousa 30 , demonstrated that α-Pinene possesses anti-inflammatory activity in mice. Another study by Bayala et al. 31 revealed that essential oils possess anti-inflammatory activity in animal models. A study by Zou et al. 32 revealed that borneol possesses significant anti-inflammatory activity. The results of GC-MS similarly revealed the presence of Alpha-terpineol. According to a study by Soleimani et al. 33 , Alpha-terpineol possesses anti-inflammatory activity in rat models.
The results of the GC-MS also revealed the presence of terpinolene. A study by Aggarwal et al. 34 , has shown that terpinolene isolated from turmeric has an anti-inflammatory effect on formalin-induced arthritis, cotton pellet granuloma and granuloma pouch models of inflammation in rats. The results of GC-MS analysis also revealed the presence of α-phellandrene. α-phellandrene is a monoterpene that is part of the essential oils found in many plants. Additionally, Gbenou et al. 35 , demonstrated that Cymbopogon citratus and Eucalyptus citriodora contained essential oils that possess significant anti-inflammatory activity by reducing formol-induced paw edema in rats. Similarly, a review by Arruda et al. 36 also reports the anti-inflammatory potential of α-phellandrene. Further, the GC-MS results revealed the presence of Limonene. In a study by Li et al. 37 , on in vitro anti-inflammatory activity of Spondias pinnata (Anacardiaceae) on Nitric oxide production, indicated that Limonene possesses anti-inflammatory activity. Studies by Vieira et al. 38 , Rufino V. 39 , Khodabakhsh et al. 40 , also demonstrate that Limonene has great potential in the management of inflammation. According to a study by Jin et al. 41 , on the phytochemical composition of the cannabis plant and its use for medicinal purposes, it is reported that Aromadendrene and terpenoids possess anti-inflammatory effects.

Limitations
This study focused on in vivo anti-inflammatory effects of E. globulus (Labill) and S. didymobotrya (Fresenius) in mice and did not explore in vitro anti-inflammatory activity of these plant extracts. Secondly, extracts of other plant parts like roots and stems were not studied for comparison purposes. Further, the extraction solvent used in this study was Dichloromethane, a non-polar solvent. Use of a polar extraction solvent may probably yield superior results.

Conclusion
This study has demonstrated that the dichloromethane leaf extracts of E. globulus (Labill) and S. didymobotrya (Fresenius) have anti-inflammatory activity in mice, which can be attributed to the constituent phytochemicals. This study confirms the traditional claim of anti-inflammatory activities of the two plants.