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International Journal of Phytomedicine and Phytotherapy

• Original contribution
• Open access
• Published: 16 January 2021

Evaluation of anti-arthritic and anti-inflammatory activities of Martynia annua L. Ethanolic extract

• Suruj Kaushik 1 ,
• Parag Jain 2 ,
• Trilochan Satapathy 1 ,
• Prerna Purabiya 1 &
• Amit Roy 1  

Clinical Phytoscience volume  7 , Article number:  7 ( 2021 ) Cite this article

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Arthritis is a disorder of change in joint architecture and ligament degeneration. Rheumatoid arthritis is an autoimmune disorder in which body’s immune system targets own cells and degrade them. Martynia annua L. has been used in Indian traditional therapies for the treatment of epilepsy, many types of inflammations, respiratory infection, sore throat and wound. The objective of the present study was to determine anti-inflammatory and anti-arthritic activities of M. annua ethanolic fruit extract.

The extraction was performed using ethanol as a solvent followed by phytochemical investigation of M. annua ethanolic fruit extract. Antioxidant and anti-hyaluronidase enzyme inhibition activities was performed for the fruit extract. In vivo anti-inflammatory of fruit extract was performed on Calotropis procera latex (CPL) induced paw edema in rats using ibuprofen as standard. Inflammation was observed at 0, 1, 2, 4 and 6 h. In vivo anti-arthritic of fruit extract was performed on Complete Freund’s adjuvant (CFA) induced arthritis in rats. Arthritis was observed at 0, 7, 14 and 21 day. X-Ray study was also performed for inflammatory and arthritic paw of rats.

The qualitative phytochemical screening of fruit extract showed presence of flavonoids, terpenoids, saponins, tannins, steroids, glycosides, proteins, carbohydrates, amino acids and polysaccharides. The antioxidant activity of fruit extract was 49.1 as compared to standard 45.73 at 100 μl dose. The anti-hyaluronidase enzyme inhibition activity of fruit extract was 84.60 as compared to standard 94.21 at 100 μl dose.

It is evident from the study that Martynia annua L. extract possess both antioxidant and hyalurinodase inhibition activity at dose dependent manner as well as anti-arthritic and anti-inflammatory potential.

Arthritis comprises varieties of joint disorders such as rheumatoid arthritis (RA), Osteoarthritis (OA) etc. those affect one joint or multiple joints [ 1 ]. Arthritis can be develop in anyone irrespective of gender, age and race [ 2 ]. The common symptoms of arthritis include swelling, tenderness and stiffness of joints as well as decrease the range of motions. It can be ranged from mild to severe conditions [ 3 ]. Person suffering from severe arthritis shows inability to walk and move as well as unable to perform daily activities [ 4 ]. It can cause permanent damage of joint architecture and degeneration of ligament [ 5 ]. RA is a systematic autoimmune disorder in which host’s immune system targets their own cells [ 6 ]. RA has been showing a global threat to many many healthy individuals. However, in India nearly 15% population i.e. 180 million people are affected from RA [ 7 ]. The prevalence of RA is higher than other diseases like cancer and diabetes. It has reported that arthritis is ranked as second most common cause of disability and considered as a significant contributor to global disability burden [ 8 ].

The incidence of RA is progresses with age and the peak is observed between the age groups of 35 and 50 years. Further the research data revealed that the globally incidence of RA is estimated as 3 in each 10,000 people [ 9 ]. The conventional treatment options for RA available in the market are analgesics, Non-steroidal anti-inflammatory drugs (NSAIDs), Disease-modifying anti-rheumatic drugs (DMARDs), and Corticosteroids. However, these drugs are accompanying with certain adverse effects such as gastro intestinal upset, ulcer and bleeding [ 10 ]. On the other hand, Phytotherapy is the oldest system of medicine in the world and it has been practiced in rural areas of India since a long time ago [ 11 ].

According to the report of World Health Organization (WHO), nearly 80% of the total population of Africa and china depend on traditional therapies and herbal based remedies [ 12 ]. Herbal based therapies occupied the highest share of the international market, with annual turnover billion dollars in Western Europe as well as in China [ 12 ]. The most frequency use of herbal medicines is due to failure of conventional therapies and their side effects [ 13 , 14 , 15 , 16 , 17 ]. Herbal medicinal drugs possess numerous qualities in the treatment of several disorders [ 18 , 19 , 20 , 21 ]. Martynia annua L. belongs to family Martyniaceae or (Pedaliaceae). It is a small, herbaceous and annual plant, distributed all over the India. It is also known as the Cat’s claw. In Ayurvedic scriptures, it is called as kakanasika, which is generally being used in Indian traditional therapies for the treatment of epilepsy, tuberculosis, sore throat and wound [ 22 ]. Interestingly, the fruits of M. annua are useful in inflammation and burns. Seeds oils are used in itching and skin affections. The fruit of M. annua is also used for its local sedative action. Thus, the primary aim of this research was to evaluate the anti-arthritic and anti-inflammatory potential of M. annua fruit extract.

Materials and methods

Chemicals and reagents.

Picric acid was purchased from Fizmerk India Chemicals, UP, India, Complete Freund’s adjuvant was procured from Sigma-Aldrich, USA, Halothane from Korten Pharmaceutical Pvt. Ltd., Ethanol and Carboxy methyl cellulose from Loba chemie, USA, Indomethacin from Jagsonpal Pharmaceuticals Pvt. Ltd. India and Ibuprofen was procured from Abbott India Ltd. All chemical and reagents use in the study were of analytical grade.

Collection, identification authentication and extraction of Martynia annua L. fruit

The fruits of M. annua were collected from village Tekari of Raipur, Chhattisgarh, India in December 2018 and authenticated by Dr. Ravindra Kumar Pandey, Professor, Dept. of Pharmacognosy; Columbia Institute of Pharmacy (CIP), Raipur, Chhattisgarh, India. The sample specimen was deposited in the herbarium of the institute wide voucher no. 0.213. The fruits were washed in running tap water; dried (360 g), crushed into coarse powder and soaked using 4.4 L of ethanol for period of 3 weeks. The extraction process with ethanol was repeated three times at room temperature by using soxhlet apparatus. The extract was distilled under reduced pressure andcontrolled temperature (40–50 °C) after extraction. The resulting semisolid residue or extract was evaporated using water bath andextract of M. annua fruit was filtered. It was then kept in an airtight dark colored container and stored in a refrigerator. The percentage yield of the extract was 19.30%.

Phytochemical analysis of M. annua fruit extract

Following tests were performed for M. annua fruit extract to determine presence of phytochemicals:

Test for carbohydrates

Molisch Test: Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. To the 2 ml of sample, 2 ml of alpha-napthol was added and mixed carefully, however, the concentrated H 2 SO 4 was added along side of the walls to the test tubes, carefully. The purple violet coloured rings were observed in between the junction of the two layers confirmed the presence of carbohydrate [ 23 , 24 ].

Test for glycosides

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. To the 1 ml of test sample solution, 3 ml of anthronone reagent was added and mixed well carefully. The formation of green coloured complex indicated the presence of glycoside [ 25 ].

Test for polysaccharides

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. To 1 ml of test sample, 2 drops of iodine solution was added. Appearance of blue coloured solution indicated the presence of polysaccharides [ 26 ].

Test for free amino acids

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. To 1 ml of test sample add 5 drops of ninhydrin and boil for 2 min. Appearance of purple coloured solution indicated the presence of amino acids [ 27 ].

Bradford’s test

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. To 0.5 ml of test sample solution, dragendroffs reagent (3 ml) was added, the appearance of blue color indicated the presence of protein [ 25 ].

Test for alkaloids

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. To 3 ml of sample dragendroff’s reagent was added and mixed well. It was then boiled for 5 mins. The appearance of dark brown or orange colour indicated the presence of alkaloids.

To 1 ml of sample Mayer’s reagent was added and mixed well carefully. It was then boiled for 5 mins. The appearance of white or pale yellow colour indicated the presence of alkaloids [ 25 ].

Test for steroids

Liberman burchard test.

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. To 2 ml of sample 10 drops of acetic acid and two drops of concentrated H 2 SO 4 were added and mixed well. Initially, red colour was observed followed by green colour that indicated the presence of steroids [ 28 ].

Salkwoski test

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. To 2 ml of sample, 2 ml of concentrated H 2 SO 4 was added and mixed vigorously. Steroids and H 2 SO 4 layers separated and sample layers forms cherry red colour and acid layer forms green colour show presence of steroids [ 28 ].

Test for triterpenes

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. To 2 ml of test sample, chloroform and concentrated H 2 SO 4 were added and mixed well. The appearance of red color indicated the presence of triterpenes [ 29 ].

Test for flavonoids

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. To 1 ml of sample 2 ml of H 2 SO 4 was added, mixed well. The appearance of yellow colour indicated the presence of flavonoids [ 30 ].

Shinoda test

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. To the ethanolic extract sample (2 ml), 5 ml of 95% ethanol was added along with few drops of conc. HCl. To this solution 0.5 g of magnesium turnings were added. The appearance of pink colour indicated presence of flavonoids [ 31 ].

Tests for tannins

Fecl 3 solution test.

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. To the 2 ml of test sample, 5% FeCl 3 Solution was added and mixed well. The appearance of deep blue black color indicated presence of tannins [ 32 ].

Dil. HNO 3 test

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. To the 2 ml of test sample, small quantity of HNO 3 Solution was added and mixed well. The appearance of reddish color indicated the presence of tannins [ 32 ].

Test for lipid

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. To 2 ml of test sample, iodine solution was added drop wise. The disappearance of of iodine color indicated the presence of lipids [ 33 ].

Test for oils

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. The 1 drop of sample was placed on a filter paper and allowed to dry. The formation of clear greasy spot indicated the presence of oils [ 34 ].

Test for saponins

Test sample was prepared by dissolving 1 g of dried ethanolic extract of M. annua with 10 ml of water. Few drops of sample was heated with alcoholic KOH and then boiled for 1 min followed by cooling. It was then acidified with 1 ml of conc. HCl. Further, a portion of it was treated with 10 ml of water and 5% NaOH was added drop wise. The formation of clear soap indicated the presence of saponins [ 32 ].

Total antioxidant capacity

The total antioxidant capacity of M. annua was determined by taking various concentrations of samples i.e. 10 μg, 50 μg and 100 μg in a clean and dry test tubes. To the test tubes, 1.9 mL of reagent solution containing 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate were added. The tubes were then subjected for incubation at 95 °C for a time period of 90 min then allowed to cool. The absorbance of the aqueous solution was observed at 695 nm against blank. Antioxidant capacities of test samples were expressed as equivalents of ascorbic acid. Ascorbic acid equivalents were calculated by using standard graph of ascorbic acid. Butylated hydroxy anisole was used as reference standard in this experiment. The values were expressed as ascorbic acid equivalents in μg per mg of extract [ 35 ].

Hyaluronidase inhibition activity

In this assay medium consisting of 3–5 U hyaluronidase, 100 μl of 20 mM sodium phosphate buffer (pH 7.0), 77 mM sodium chloride, 0.01% Bovine Serum Albumin were added. The mixture was pre-incubated with various concentrations such as 10 μg, 50 μg and 100 μg of the test compound for 15 min time period by maintaining temperature at 37 °C. The assay was commenced after addition of 100 μl hyaluronic acid and0.03% of 300 mM sodium phosphate (pH 5.35) to the incubation mixture. It was then allowed to incubate again for 45 min at temperature 37 °C. However, the undigested hyaluronic acid was precipitated with 1 ml acid albumin solution made up of 0.1% bovine serum albumin in 24 mM sodium acetate and 79 mM acetic acid (pH 3.75). The absorbance of the reaction mixture was measured at 600 nm after standing the reaction mixture at room temperature for a period of 10 min. The absorbance in the absence of enzyme was used as the reference standard for maximum inhibition. The inhibitory potential of M. annua fruit extract was calculated as the percentage ratio of the absorbance in the presence of test compound vs. absorbance in the absence of enzyme. The enzymatic activity was measured by control experiment run simultaneously in which the enzyme was pre-incubated with 5 μl DMSO instead, and followed by the assay procedures described above. Indomethacin was used as a reference drug in this experiment [ 36 ].

In vivo anti-inflammatory and anti-arthritic activities

Experiment animals.

Albino wistar rats weighing between 150 and 200 g of both sex were procured from Institutional animal house facility, Columbia Institute of Pharmacy, Raipur (C.G.). The protocol was reviewed by the expert committee (IAEC), considered and approved (Approval No is CIP/IAEC/2017/103 and Regd.No.1321/PO/ReBi/S/10/CPCSEA, Dated 22/10/2014). The animals were allowed for acclimatization for a period of two weeks before starting of the experiments. They were housed in the polypropylene cages with husk bedding. Standard pelleted diet and aqua guard water were provided to the animals during entire experimental duration. All animals were maintained with controlled environment, 12 h light/ 12 h dark cycle and relative humidity were maintained according to CPCSEA guidelines. The animals were examined at regular intervals for behavioral abnormalities if any.

Acute toxicity study

The toxicity potential of the test compound was evaluated according to OECD guideline 423.

Screenings of anti-inflammatory activity

Calotropis procera latex (cpl) induced paw edema in rats.

Calotropis procera latex (CPL) was collected from tissues of plant stem by making an incision. CPL was centrifuged at 5000×g and supernatant was isolated as sticky rubber like matter. It was dried for 2–3 days and then triturated in a mortar pestle with small amount of water to get the ready aqueous suspension of CPL for parenteral administration [ 37 ].

Albino wistar rats weighing 150-200 g were selected for the study. They were divided into five groups each containing six animals. Group 1 was negative control group, received vehicle p.o.; Group 2 was positive control group, received 0.1 ml of CPL injected on sub-plantar surface of right hind paw; Group 3 and Group 4 were test groups received 200 and 400 mg/kg plant extract, respectively; and Group 5 was reference group, received ibuprofen (72 mg/kg). All rats were injected with 0.1 ml of CPL in normal saline into sub planter area of right hind paw. Test substance and standard drug were administered 1 h prior to CPL injection [ 38 ].

Complete Freund’s adjuvant (CFA) induced arthritis in rats

Albino wistar rats weighing 150-200 g were selected for the study. They were divided into five groups each containing six animals. Group 1 was negative control group, received vehicle p.o.; Group 2 was positive control group, received 0.1 ml of CFA emulsion injected on sub-plantar surface of right hind paw; Group 3 and Group 4 were test groups received 200 and 400 mg/kg plant extract, respectively; and Group 5 was reference group, received indomethacin (10 mg/kg). Arthritis was induced by sub-plantar injection of 0.1 ml of CFA on right hind paw of rats [ 39 ].

Assessment of inflammation and arthritis

Test drug was administered p.o. once a day from the day of injection of induction and continued up to 14 days after treatment. The assessment of inflammation and arthritis was done on following parameters:

Paw volume: The change in the inflammatory reaction was measured using mercury plethysmograph on 0, 7, 14, and 21 day from the day of induction.

Body Weight: The change in the body weight was calculated using digital weighing balance on 0, 7, 14, and 21 day from the day of induction.

X-ray study of arthritic paw of rats

X-ray study of paw was performed to determine the protective effect of M. annua fruit extract at the completion of dosing schedule. The two animals from each groups in CFA induced arthritis model were selected for X-ray examination. The animals were anesthetized with halothane before X-ray examination and then paw was placed on the smooth surface to take X-ray photograph using portable digital X-ray apparatus [ 40 ].

Statistical analysis

The data were expressed as mean ± SEM for each of the parameters studied and were analyzed using One-Way ANOVA by Graph pad INSTAT, and Post hock analysis were done followed by Dunnet’s test. P  < 0.05 was considered to be statistically significant.

Phytochemical analysis

The result of qualitative Phytochemical screening of fruit extracts of M. annua are represented in Table 1 . Results showed the presence of several secondary metabolites such as flavonoids, tannins, steroids, terpenoids, saponins, glycosides, Proteins, carbohydrates, amino acids, and polysaccharides in plant extract. However, flavonoids and tannins were the major groups of compounds that act as primary antioxidants or free radical scavengers.

Table 2 representing the total anti oxidant capacity of the M. annua fruit extract at different concentrations i.e. 10 μl, 50 μl and 100 μl. The extract at 100 μl showing total antioxidant activity i.e. 49.1 as compared to standard BHA i.e. 45.73. The antioxidant capacities were expressed as equivalents of ascorbic acid and Butylated hydroxy anisole (BHA) which was used as a reference standard. The values were expressed as ascorbic acid equivalents in μg per mg of extract. The absorbance of the aqueous solution of each sample was measured at 695 nm against a blank. This analysis showed an increase in total antioxidant capacity with increase in concentration of the extract.

Effect of M. annua on hyaluronidase enzyme inhibition activity

Table 3 shows the hyalurinodase inhibitory activity of M. annua fruit extract. The percentage inhibition of hyalurinodase enzyme by the extract at concentration of 50 μg was found to be 73.66, however, at 100 μg the hyalurinodase inhibition was 84.6 in comparison to the control group. The result revealed that the fruit extract of M. annua at different concentration (10 μl, 50 μl and 100 μl) exhibited dose dependent hyalurinodase inhibition and thus, proved to be effective as an anti-inflammatory agent.

The ethanolic extract of M. annua was found non-toxic to the animals upto 2000 mg/kg dose. No any sign of toxicity in terms of change of fur color, behavioral change, writhing response, lethargy, change in urination and feeding habits were seen upto 14 days of observation.

In vivo anti inflammatory activity (CPL induced paw edema)

In-vivo anti-inflammatory activity of M. annua extract was carried out using CPL induced paw edema model in rats. Effect of extract on CPL induced paw edema in rats is depicted in Table 4 and Fig. 1 . Results exhibited that M. annua extract possesses better anti inflammatory activity at high concentration than its lower dose whereas standard drug ibuprofen exhibit more significant anti-inflammatory activity than M. annua treated groups. Results exhibited that M. annua significantly reduced inflammation in paw at higher dose in comparison to the control group.

Effect of Martynia annua L. extract on CPL induced paw edema in rats. a  = Control group, b = Low dose group (200 mg/kg), c = High dose group (400 mg/kg), d  = Standard

In vivo antiarthritic activity (CFA induced arthritis)

The arthritis in animals was induced by injection of CFA intra articularly. Effect of M. annua extract on CFA induced paw edema in rats is shown in Table 5 and Fig. 2 . The results of antiarthritic activity revealed that the extract significantly reduced arthritis in animals i.e. 64.28 in comparison to the control group. However, reduction in arthritis was maximum by standard group.

Effect of Martynia annua L. extract on CFA induced paw edema in rats. a  = Control group, b  = Low dose group (200 mg/kg), c  = High dose group (400 mg/kg), d  = Standard

X-ray assessment of inflamed paw of rats

The X-ray result revealed that the animals treated with high concentration of test compound showing more protection of the cartilages in compared to low concentration. Hence, it has proved that the extract produced dose depended protection against CPL and CFA induced arthritis as shown in Fig. 3 and Fig. 4 , respectively.

X-ray images of CPL induced paw edema in rats. a  = Control group, b  = Low dose group (200 mg/kg), c = High dose group (400 mg/kg), d  = Standard

X-ray images of CFA induced paw edema in rats. a  = Control group, b  = Low dose group (200 mg/kg), c  = High dose group (400 mg/kg), d  = Standard

The present study was performed to carryout the phytochemical screening of the dried fruit extracts of M. annua and to evaluate its in vitro and in vivo pharmacological activities. Herbal drugs possess several active constituents that are responsible for anti-inflammatory activity [ 41 , 42 ]. The qualitative analysis of the sample exhibited the presence of secondary metabolites such as flavonoids, alkaloids, steroids, tannins, saponins and glycosides. Similar compounds were identified by Muazzam et al., 2018 in their study [ 43 ]. The fruit extract also exhibited antioxidant potential. The ethanolic extract showed maximum extent of total antioxidant activity as well as hyalurinodase inhibition. The phenolic compounds and flavonoids were believed to be responsible of antioxidant activities. Our results were in agreement with the findings of Arshad et al., 2017 [ 44 ]. The researchers reported the antioxidant activity of M. annua fruit extract was dose dependent. Herbal drugs are more safer than the synthetic drugs due to several toxicities associated with them [ 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 ]. Similarly in case of hyalurinodase inhibition assay, the extract was screened at three different concentrations such as 10 μl, 50 μl and 100 μl. To evaluate the anti inflammatory potential of the M. annua extract at different concentrations CPL induced paw edema model in rats was selected. The result of anti inflammatory potential of extract exhibited that the extract at higher concentration possess maximum reduction in paw edema at 6 h i.e. 65.33, where as the standard drug ibuprofen exhibited 60.08 at 6 h. The arthritis in animals was induced by injection of CFA intra-articularly. The result of anti-arthritic activity revealed that theextract at higher concentration exhibited maximum protection i.e. 64.28 as compared to control group. The anti-inflammatory and anti arthritic activities of herbal extract in our study were also supported by the work of Foyet et al., 2014 based on their study on Vitellaria paradoxa stem bark extract against inflammation and arthritis [ 53 ]. Chandel et al., 2013, reported that fruit extract of plants possess significant anti-arthritic activity [ 54 ].

It has evident from the study that M. annua extract possess both antioxidant and hyalurinodase inhibition activity at dose dependent manner as well as anti-arthritic and anti-inflammatory potential. Further, research at molecular level as well as clinical study using human volunteers will pave the way to establish safety and effectiveness of such extract there by it can be formulated into suitable dosage form commercialization for the overall well being of the society.

Availability of data and materials

It can be made available on request.

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Kaushik, S., Jain, P., Satapathy, T. et al. Evaluation of anti-arthritic and anti-inflammatory activities of Martynia annua L. Ethanolic extract. Clin Phytosci 7 , 7 (2021). https://doi.org/10.1186/s40816-021-00250-y

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• Martynia annua
• Inflammation
• Calotropis procera
• Hyaluronidase

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Research developments in the syntheses, anti-inflammatory activities and structure–activity relationships of pyrimidines †.

* Corresponding authors

a Institute of Chemistry, Federal University of Mato Grosso do Sul, Campo Grande, MS, Brazil E-mail: [email protected]

b Department of Chemistry, Sarhad University of Science and Information Technology, Peshawar, Khyber Pakhtunkhwa, Pakistan

c Department of Chemistry, Islamia College University, Peshawar, Khyber Pakhtunkhwa, Pakistan

d Department of Chemistry, University of Malakand, Chakdara, Dir (L), Khyber Pakhtunkhwa, Pakistan E-mail: [email protected]

Pyrimidines are aromatic heterocyclic compounds that contain two nitrogen atoms at positions 1 and 3 of the six-membered ring. Numerous natural and synthetic pyrimidines are known to exist. They display a range of pharmacological effects including antioxidants, antibacterial, antiviral, antifungal, antituberculosis, and anti-inflammatory. This review sums up recent developments in the synthesis, anti-inflammatory effects, and structure–activity relationships (SARs) of pyrimidine derivatives. Numerous methods for the synthesis of pyrimidines are described. Anti-inflammatory effects of pyrimidines are attributed to their inhibitory response versus the expression and activities of certain vital inflammatory mediators namely prostaglandin E 2 , inducible nitric oxide synthase, tumor necrosis factor-α, nuclear factor κB, leukotrienes, and some interleukins. Literature studies reveal that a large number of pyrimidines exhibit potent anti-inflammatory effects. SARs of numerous pyrimidines have been discussed in detail. Several possible research guidelines and suggestions for the development of new pyrimidines as anti-inflammatory agents are also given. Detailed SAR analysis and prospects together provide clues for the synthesis of novel pyrimidine analogs possessing enhanced anti-inflammatory activities with minimum toxicity.

• This article is part of the themed collections: A celebration of Latin American research in RSC Advances and 2021 Reviews in RSC Advances

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Research developments in the syntheses, anti-inflammatory activities and structure–activity relationships of pyrimidines

H. U. Rashid, M. A. U. Martines, A. P. Duarte, J. Jorge, S. Rasool, R. Muhammad, N. Ahmad and M. N. Umar, RSC Adv. , 2021,  11 , 6060 DOI: 10.1039/D0RA10657G

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The anti-inflammatory and antioxidant activity of 25 plant species used traditionally to treat pain in southern African

• Salmon A. Adebayo 1 , 2 ,
• Jean P. Dzoyem 1 , 3 ,
• Leshweni J. Shai 2 &
• Jacobus N. Eloff 1  

BMC Complementary and Alternative Medicine volume  15 , Article number:  159 ( 2015 ) Cite this article

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Inflammation is a common risk factor in the pathogenesis of conditions such as infections, arthritis, type 2 diabetes mellitus, obesity and cancer. An ethnobotanical survey of medicinal plants used traditionally to treat inflammation and related disorders such as pain, arthritis and stomach aches in southern Africa led to the selection of 25 plant species used in this study.

The antioxidant activities of acetone extracts were determined by measuring the free radical scavenging activity and ferric reducing ability, respectively. The anti-inflammatory activities of the extracts were determined by measuring the inhibitory effect of the extracts on the activities of the pro-inflammatory enzyme, lipoxygenase and inducible nitric oxide synthase.

Extracts of Peltophorum africanum had good antioxidant activity with IC 50 values of 4.67 ± 0.31 μg/mL and 7.71 ± 0.36 μg/mL compared to that of the positive control ascorbic acid (2.92 ± 0.14 μg/mL and 13.57 ± 0.44 μg/mL), using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging and 2,2′-azinobis (3-ethylbenzthiazoline-6-sulphonic acid (ABTS) methods, respectively. The metabolism of linoleic acid to leukotriene derivatives by 15-lipoxygenase (15-LOX) was also inhibited by the crude acetone extracts of Peltophorum africanum (IC 50  = 12.42 μg/mL), Zanthoxylum capense (IC 50  = 14.92 μg/mL) compared to the positive control quercetin (IC 50  = 8.75 μg/mL). There was a poor correlation between the flavonoid content and 15-LOX inhibition by the extracts (R 2  = 0.05), indicating that flavonoids are not involved in LOX inhibition. Extracts of Clausena anisata, at a concentration of 6.25 μg/mL inhibited nitric oxide production by RAW 264.7 macrophage cell lines in vitro by 96 %. The extracts of Zanthoxylum capense were the least cytotoxic (IC 50  > 1000 μg/mL) when the extract toxicity was determined against Vero (African green Monkey) kidney cell lines.

Some plant species used traditionally to treat pain have reasonable anti-inflammatory activity and flavonoids are probably not involved in this process.

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Medicinal plants have long been recognised as important sources of therapeutically active compounds. Evidence-based research supports the medical and pharmacological benefits of plant-derived compounds, with increasing interest in the identification and characterization of bioactive compounds from natural sources [ 1 ].

One of the earliest recorded approaches for treating inflammation and pain was the application of extracts from willow leaves by Celsius in 30 AD [ 2 ]. This observation led to the discovery of acetyl salicylic acid, the active component of aspirin, a major anti-inflammatory drug widely used in clinical practice, along with many other non-steroidal anti-inflammatory drugs (NSAIDs) in current use [ 3 ].

Non-steroidal anti-inflammatory drugs are commonly prescribed for treatment of pain and inflammatory conditions such as rheumatoid arthritis, osteoporosis and Alzheimer’s disease. However, because many NSAIDs are associated with side effects such as gastrointestinal bleeding and suppressed function of the immune system [ 4 ], attention has shifted to alternative pharmacotherapies [ 5 , 6 ]. Recent studies on Zingiber officinale , ginger, suggest that it might be as effective as some NSAIDs in the treatment of inflammation and related pain [ 7 , 8 ].

In South Africa the use of plants to treat many diseases is widely practiced. According to Iwalewa et al. [ 9 ], more than 115 plant species of 60 families are used in South Africa to treat pain-related inflammatory disorders in humans and animals. The bioactive principles in these plant species have been linked to secondary metabolites such as phenolic compounds (curcumins, flavonoids and tannins), saponins, terpenoids and alkaloids [9, 10,]. Biological and therapeutic properties attributed to these plant metabolites include antioxidant, anti-inflammatory, antimicrobial and anticancer activities [ 10 ]. The mechanisms of action of many phenolic compounds such as flavonoids, tannins and curcumins are thought to be via their free radical scavenging activities or the inhibition of pro-inflammatory enzymes such as cyclo-oxygenases (COX) and lipoxygenases (LOX) in the inflammatory cascades [ 11 , 12 ].

Flavonoids are a group of polyphenols thought to inhibit the biosynthesis of prostaglandins, end-products in the COX and LOX pathways of immunologic responses [ 13 ]. There are three known isomeric-forms of COX i.e. COX-1 and COX-2, with a recently described third isomeric-form, COX-3 that is selectively inhibited by acetaminophen and related compounds [ 14 , 15 ]. The selective inhibition of COX-2 is more desirable because the inhibition of COX-1 in the gastric mucosa is associated with the undesirable effects of NSAIDs [ 16 ]. COX-2 is induced as an early response to pro-inflammatory mediators and stimuli such as endotoxins and cytokines [ 17 ]. Upon induction, COX-2 synthesizes prostaglandins that contribute to inflammation, swelling and pain [ 18 ]. Consequently, dual COX-2/LOX inhibitor compounds could potentially be developed into safer and more effective drugs for the treatment of inflammation since they could potentially inhibit biosynthesis of prostaglandins and leukotrienes respectively from arachidonic acid [ 16 , 19 ], without the undesirable effects of NSAIDs.

Lipoxygenases are lipid-peroxidizing enzymes involved in the biosynthesis of leukotriene from arachidonic acid, mediators of inflammatory and allergic reactions. These enzymes catalyse the addition of molecular oxygen to unsaturated fatty acids such as linoleic and arachidonic acids [ 20 ]. There are four main iso-enzymes already described, namely, 5-LOX, 8-LOX, 12-LOX and 15-LOX, depending on the site of oxidation in the unsaturated fatty acids [ 20 ]. The common substrates for LOX are linoleic and arachidonic acids. For many in vitro studies, soy bean LOX is used due to difficulties in obtaining human LOX for bioassays [ 21 ].

During inflammation, arachidonic acid is metabolized via the COX pathway to produce prostaglandins and thromboxane A 2 , or via the LOX pathway to produce hydroperoxy-eicosatetraenoic acids and leukotrienes [ 22 ]. The LOX pathway is active in leucocytes and many immune-competent cells including mast cells, neutrophils, eosinophils, monocytes and basophils. Upon cell activation, arachidonic acid is cleaved from cell membrane phospholipids by phospholipase A 2 and donated by LOX activating protein to LOX, which then metabolises arachidonic acids in a series of reactions to leukotrienes, a group of inflammatory mediators [ 23 ]. Leukotrienes act as phagocyte chemo-attractant, recruiting cells of the innate immune system to sites of inflammation. For instance in an asthmatic attack, it is the production of leukotrienes by LOX that causes the constriction of bronchioles leading to bronchospasm [ 8 , 16 ]. Therefore, the selective inhibition of LOX is an important therapeutic strategy for asthma [ 8 , 16 , 24 ]. Inhibitors of the activities of LOX could provide potential therapies to manage many inflammatory and allergic responses. Medicinal plants may therefore be potential sources of inhibitors of COX-2/LOX that may have fewer side effects than NSAIDs [ 24 ].

Nitric oxide (NO) is a short-lived free radical that mediates many biological processes. One of the functions of NO is to enhance the bactericidal and tumoricidal activities of activated macrophages [ 25 , 26 ]. Excessive production of NO could however potentially lead to tissue damage and activation of pro-inflammatory mediators [ 27 , 28 ]. The potential of extracts from medicinal plants to scavenge these free radicals and modulate inflammatory reactions has been demonstrated [ 29 – 31 ].

The objective of this study was to determine the anti-inflammatory activity of extracts in relevant bioassays in order to validate their use for pain relief and to identify plants that could be investigated in more detail.

Analytical grade chemicals were purchased from various suppliers in South Africa, and were used for the bioassays in the laboratory.

Preparation of plant materials

Fresh leaves of the selected plants species were collected from the Manie van der Schijff Botanical Garden, University of Pretoria in March 2012. The plant materials were dried at room temperature in a well-ventilated room for a week. After drying, the materials were ground to fine powder using a MacSalab Model 200 grinder and stored in closed honey jars in the dark. Herbarium specimens for each of the plant species were prepared and deposited at HGWJ Schweickerdt Herbarium, University of Pretoria. Herbarium voucher specimen numbers (PRU voucher numbers) are provided in Table  1 .

Preparation of crude extracts for biological assays

Ground leaf powders (3 g) were extracted in 30 mL of 70 % acetone in clean honey jars and vigorously shaken for 3 h (Labotec model 20.2 shaker). The crude acetone extracts were filtered through Whatman No. 1 filter papers into pre-weighed honey jars, and then left open overnight for solvent evaporation. The honey jars were weighed again to determine the percentage yield of the crude extracts. For the biological assays, the crude extracts were reconstituted in dimethyl sulphoxide (DMSO) at a concentration of 10 mg/mL.

Determination of total phenolics and flavonoids

Total phenolics were determined according to the method of Folin-Ciocalteu described by Makkar [ 32 ], with slight amendments. In brief, 25 μL of crude extract was treated with 250 μL Folin-Ciocalteu reagent for 5 min. The reaction was stopped by adding 750 μL 20 % anhydrous sodium carbonate. The volume was made up to 5 mL with distilled water and incubated in the dark at room temperature for 2 h. After incubation, the absorbance was read at 760 nm with a spectrophotometer (HELIOS βT60, Separation Scientific). The phenolic content was determined from a standard curve of different concentrations of gallic acid DMSO. The results were expressed as mg/g gallic acid equivalent (GAE).

Flavonoid content of the extracts was determined using the methods of Yadav and Agarwala, [ 33 ], also amended slightly. Crude extracts (100 μL) were dissolved in 300 μL methanol, to which 20 μL 10 % aluminium chloride was added. A further 20 μL of 1 M sodium acetate was added to the solution. The resultant solution was made up to 1 mL with distilled water. This was incubated at room temperature for 30 min in a microplate. After incubation, the absorbance was read at 450 nm in a microplate reader (SpectraMax 190, Molecular devices). Quercetin (10 mM) was used as a standard. The flavonoid content of each extract was expressed as mg/g quercetin equivalent (QE).

The 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay methods

The DPPH radical-scavenging activity was determined using the method of Brand-Williams et al. [ 34 ]. Ascorbic acid and Trolox were used as positive controls, methanol as negative control and extract without DPPH as blank. Results were expressed as percentage reduction of the initial DPPH absorption in relation to the control. The concentration of extract leading to 50 % reduction of DPPH (IC 50 ) was also determined.

The 2, 2′-azinobis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging assay methods

The ABTS radical scavenging capacity of the samples was measured with modifications of the 96-well microtitre plate method described by Re et al. [ 35 ]. Trolox and ascorbic acid were used as positive controls, methanol as negative control and extract without ABTS as blank. The percentage of ABTS• + inhibition was calculated using the formula:

where OD represents the optical density or absorbance.

The IC 50 values were calculated from the graph plotted as inhibition percentage against the concentration.

The ferric reducing ability of plasma (FRAP) assay methods

The FRAP assay was carried out according to the procedure of Benzie and Strain [ 36 ] with slight modification. The FRAP assay depends upon the reduction of ferric tripyridyltriazine (Fe (III)-TPTZ) reduction to ferrous tripyridyltriazine (Fe (II)-TPTZ) by a reductant at low pH. Ferrous (II)-TPTZ has an intensive blue colour and can be monitored at 593 nm. Briefly, the FRAP reagent was prepared using an acetate buffer (pH 3.6), 10 mM TPTZ solution in 40 mM hydrochloric acid and 20 mM iron (III) chloride solution in proportions of 10:1:1 (v/v), respectively. Twenty five microliters of sample were added to 175 μL of the FRAP reagent. The absorbance of the reaction mixture was recorded at 593 nm (SpectraMax 190, Molecular devices) after 5 min. The standard curve was made using iron (II) sulphate solution (40–0.078 μg/mL), and the results were expressed as μg Fe (II)/g of extract. All the measurements were taken in triplicate and the mean values were calculated.

Inhibition of 15-lipoxygenase (15-LOX) enzyme

The 15-LOX (Sigma) was made up to a working solution of 200 units/mL and kept on ice. A volume of 12.5 μL of test sample or control (dissolved in DMSO) was added to 487.5 μL of 15-LOX in a 96-well microtitre plate and incubated at room temperature for 5 min. After incubation, 500 μL substrate solutions (10 μL linoleic acid dissolved in 30 μL ethanol, made up to 120 mL with 2 M borate buffer at pH 9.0) was added to the solution. After 5 min incubation at room temperature, the absorbance was measured with the microplate reader at 234 nm (SpectraMax 190, Molecular devices). Quercetin (1 mg/mL) was used as a positive control, while DMSO was used as the negative control (100 % enzyme activity or no enzyme inhibition). The percentage enzyme inhibition of each extract compared with negative control as 100 % enzyme activity was calculated using the equation;

The results were expressed as IC 50, i.e. concentration of the extracts and controls that resulted in 50 % 15-LOX inhibition plotted on a graph.

Inhibition of nitric oxide (NO) production

Cell culture.

The RAW 264.7 macrophage cell lines obtained from the American Type Culture Collection (Rockville, MD, USA) were cultured in plastic culture flasks in Dulbecco’s Modified Eagle’s Medium (DMEM) containing l-glutamine supplemented with 10 % foetal calf serum (FCS) and 1 % PSF (penicillin/streptomycin/fungizone) solution under 5 % CO 2 at 37 °C, and were split twice a week. Cells were seeded in 96 well-microtitre plates and were activated by incubation in medium containing LPS (5 μg/mL) and various concentrations of extracts dissolved DMSO.

Measurement of nitrite

Nitric oxide released from macrophages was assessed by the determination of nitrite concentration in culture supernatant using the Griess reagent. After 24 h incubation, 100 μL of supernatant from each well of cell culture plates was transferred into 96-well microtitre plates and equal volume of Griess reagent was added. The absorbance of the resultant solutions in the wells of the microtitre plate was determined with a microtitre plate reader (SpectaMax 190 Molecular devices) after 10 min at 550 nm. The concentrations of nitrite were calculated from regression analysis using serial dilutions of sodium nitrite as a standard. Percentage inhibition was calculated based on the ability of extracts to inhibit nitric oxide formation by cells compared with the control (cells in media without extracts containing triggering agents and DMSO), which was considered as 0 % inhibition.

Cell viability

To ensure that the observed nitric oxide inhibition was not due to cytotoxic effects, the cytotoxicity was also determined against Vero Monkey kidney cells as previously described by Mosmann [ 37 ], with slight modifications. After removal of media, the cells were topped up with 200 μL DMEM. To each well, 30 μL of 15 mg/mL 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetra-zoliumbromide (MTT) was added. The cells were incubated at 37 °C with 5 % CO 2 . After 2 h, the medium was carefully discarded and the formed formazan salt was dissolved in DMSO. The absorbance was read at 570 nm (SpectraMax 190, Molecular devices). The percentage of cell viability was calculated with reference to the control (cells without extracts containing LPS taken as 100 % viability).

All the experiments to measure nitric oxide inhibition were conducted three times in triplicate.

Cytotoxicity assessments

The cytotoxicity of the extracts (dissolved in acetone) against Vero monkey kidney cells was assessed by the MTT reduction assay as previously described [ 37 ] with slight modifications. Cells were seeded at a density of 1 × 10 5 cells/mL (100 μL) in 96-well microtitre plates and incubated at 37 °C and 5 % CO 2 in a humidified environment. After 24 h incubation, extracts (100 μL) at varying final concentrations were added to the wells containing cells. Doxorubicin (40–0.38 μM) was used as a reference compound. A suitable blank control with equivalent volume of acetone was also included and the plates were further incubated at 37 °C for 48 h in a CO 2 incubator. The medium was removed by aspiration and cells were then washed twice with PBS, followed by suspension in fresh medium (200 μL). Then, 30 μL of MTT (5 mg/mL in PBS) was added to each well and the plates were incubated at 37 °C for 4 h. The medium was removed by aspiration and 100 % DMSO (100 μL) added to dissolve the formed formazan crystals. The absorbance was measured on SpectraMax 190 (Molecular devices) microtitre plate reader at 570 nm. The percentage of cell growth inhibition was calculated based on a comparison with untreated cell. The selectivity index (SI) values were calculated by dividing cytotoxicity LC 50 values by the MIC values (SI = LC 50 /MIC).

Statistical analysis

All results are presented as the means of triplicate experiments. Differences between test extracts in these experiments was assessed for significance using analysis of variance (ANOVA) and student t -test, where probability (p ≤ 0.05) was considered significant.

Results and discussion

The results obtained in this study are presented below using Tables and Figures for ease of interpretation and data comparison.

Crude yield of extracts

Tulbaghia violacea yielded 22 % of crude acetone extract from 3 g plant material, the highest yield of all the plant species in this study. This plant grows as a bulbous rhizome, which had to be cut into pieces for proper drying. The presence of reserve materials might account for the high yield of extract from the plant unlike the other plant species in the study, whose leaves could be easily dried when left open in the drying room for three days (Table  1 ).

Total phenolics and flavonoid contents

The high extract yield from T. violacea did not correlate well with its total phenolics and flavonoid content. This may be due to high concentrations of carbohydrates as reserve material in the rhizome. Terminalia phanerophlebia and Terminalia prunioides with lower crude extract yield of 7 % and 5.7 % respectively contained more total phenolics than T. violacea (Table  1 ). The highest amounts of total phenolic compounds were obtained from T. phanerophlebia (86 mg/g GAE) followed by T. prunioides (79 mg/g GAE) and M. comosus (64.7 mg/g GAE).

In terms of flavonoid content, the three highest yields were obtained from D. cinerea (0.54 mg/g QE), T. phanerophlebia (0.53 mg/g QE) and S. birrea (0.52 mg/g QE), respectively (Fig.  1 ). The overall results suggest that generally, there was poor correlation between total phenolics and flavonoid contents in the selected plant species (R 2  = 0.05); however, T. phanerophlebia seems to be an exception. Not much study has been done on phyto-chemical screening of the leaves of T. phanerophlebia, but available literature data indicates the presence of triterpenoids and tannins [ 10 ]. The dried leaves of the plant are generally used as decoction in water to treat rheumatism, stomach pains and diarrhoea [ 38 ]. The high content of total phenolics and flavonoids, possibly tannins, triterpenoids and other secondary metabolites may be responsible for its therapeutic uses.

Relationship between total phenolic and flavonoid contents. Results indicated that there was no correlation between the total phenolic content and flavonoid content of the extracts tested (R 2  = 0.05)

Data from literature sources on the secondary metabolites present in the leaves of T. prunioides is scarce. Its antibacterial [ 39 ], Thin Layer Chromatography profile and antifungal activity [ 40 ], and antioxidant activity [ 41 ] has been reported. However, the dried leaves are used as decoction traditionally for the relief of stomach pains. Our study indicated that it contained relatively high amounts of phenolic compounds, possibly flavonoids, tannins and terpenoids, this may be responsible for the antimicrobial and antioxidant activity. The third plant species with a high phenolic content among the selected plants was M. comosus. Potential anti-fungal and lipoxygenase inhibitory properties of this plant species have already been reported. This may be associated with its flavonoid and cardiac glycoside content [ 42 ]. Phenolic compounds, especially flavonoids are well known for their anti-oxidant activitiy and lipoxygenase enzyme inhibitory activity [ 43 ].

Anti-inflammatory activities

The main objective of the study was to evaluate the anti-inflammatory activity of the selected extracts using the anti-15 LOX model of inhibition. Therefore the three plants extracts with promising inhibitory activity of 15-LOX were selected for further investigation. As illustrated in Fig.  2 , crude extracts harvested from two of the plant species tested, P. africanum (IC 50  = 12.42 μg/mL) and Z. capense (IC 50  = 14.92 μg/mL), had promising 15-LOX inhibitory activities compared with quercetin (IC 50  = 8.75 μg/mL) used as a positive control. These complex crude extracts may contain compounds with higher activity than quercetin. These results suggest that the bioactive constituent(s) of P. africanum had both antioxidant and anti-inflammatory activities. Antioxidants act by scavenging free radicals such as reactive oxygen species, hydroxyl radicals and nitric oxide while anti-inflammatory mediators act by modulating the activities of pro-inflammatory enzymes and cytokines.

Inhibitory activities of crude plant extracts on 15-LOX. The extract with the highest inhibitory activity on 15-LOX was obtained from P. africanum (IC 50  = 12.42 μg/mL) compared with quercetin controls (IC 50  = 8.75 μg/mL)

The lipoxygenase group of enzymes (5, 8, 12 and 15-LOX) plays a role in many inflammatory disorders. The isomeric enzyme, 15-LOX is an important enzyme involved in the synthesis of leukotrienes from arachidonic acids. Biologically active leukotrienes are mediators of many pro-inflammatory and allergic reactions, therefore the inhibition of the synthesis of leukotrienes by 15-LOX is considered as one of the therapeutic strategies in the management of inflammatory conditions [ 17 , 24 ]. Assessment of extracts derived from more than 180 different plant species indicated their potential dual COX/LOX inhibitory capacity [ 24 ]. Extracts or compounds from plants inhibiting the pro-inflammatory activities of these enzymes may contain potential leads or templates for the development of potent anti-inflammatory drugs [ 44 ]. Further work is required to properly characterize the compound(s) responsible for the anti-inflammatory principles in these plant species, and also understand their mechanisms of action. The three plants extracts with promising inhibitory activity on 15-LOX were selected for further investigation.

Inhibition of nitric oxide (NO) production by the three extracts with promising 15-LOX inhibitory activity

The inhibitory activity of the extracts on NO production by induced RAW 264.7 macrophage cell lines is presented in Table  2 . Clausena anisata crude acetone extracts had the best inhibitory activity on NO production (96.9 % inhibition/ 71.3 % cell viability) at 6.25 μg/mL compared with P. africanum (91.3 %/ 65.7 %), Z. capense (62.2 %/ 82.5 %) and quercetin (91.1 %/ 73.8 %) respectively (Table  2 ). Extracts with good inhibitory activity on NO production and a low cytotoxicity are more useful. Release of NO promotes inflammation, therefore extracts that could act as scavengers of NO, or inhibitors of its production, especially with corresponding low cytoxicity could be used to mitigate the propagation of inflammation by NO. The inhibition of NO production by extracts derived from medicinal plants may be due to the inhibition of inducible nitric oxide synthase activity and/or its expression [ 30 , 31 ].

Antioxidant activities

Phytochemical evaluation of extracts derived from P. africanum has yielded bergenin [ 45 ] and betulinic acid [ 46 ]. Secondary metabolites are stored in various parts of the plant; however coumarins constitute the major compounds in the leaves [ 47 ]. The plant is widely used traditionally for treating wounds, back and joint pains and dysentery, among others [ 48 ], but reported biological activities of the extracts are limited to antimicrobial activity [ 49 ]. The bioactive compounds responsible for the observed effects have not been properly characterized and the mechanism of activity has not been explored. In the case of Z. capense, biological activities such as anti-mycobacterial [ 50 ] and anti-proliferative effects [ 51 ] have been reported, subsequent to a bio-assay guided isolation of six alkaloids from the roots of the plant [ 52 ].

Extracts of P. africanum had the best antioxidant activity among the three extracts that were tested (Table  3 ). With IC 50 of 4.67 ± 0.31 and 7.71 ± 0.36 μg/mL using the DPPH and ABTS assays respectively, the results were comparable to that of Trolox positive controls (2.74 ± 0.08 and 7.21 ± 0.42 μg/mL). This is consistent with the findings of Bizimenyera, 2007 [ 53 ]. An extensive review of extracts of African medicinal plants with potent anti-oxidant activities by Atawodi [ 54 ] indicated that the mechanism(s) of action extracts was by free radical scavenging. In addition, the synergistic effects of natural products also enhance their antioxidant activities [ 53 ]. These results suggest that the bioactive constituent(s) of P. africanum had both antioxidant and anti-inflammatory activities. Antioxidants acts by scavenging free radicals such as reactive oxygen species, hydroxyl radicals and nitric oxide while anti-inflammatory mediators act by modulating the activities of pro-inflammatory enzymes and cytokines. Accumulation of free radicals result in cellular injury, and may be the cause of many diseases.

Cytotoxicity

Our results indicated that extracts of Z. capense had the lowest cytotoxicity on Vero Monkey kidney cell lines (Table  3 ) among those tested (IC 50  > 1000 μg/mL). Peltophorum africanum extracts also had a relatively low toxicity of with an IC 50 of 103 μg/mL that was comparable to values in an earlier report [ 53 ]. The safety of herbal remedies remains a concern because few reports exist on the safe use of these products. Many extracts have been shown to contain potentially harmful substances that could impact adversely on human health when consumed [ 55 ]. Although, our study suggests that extracts of Z. capense had low toxicity on Vero cell lines (≥1000 μg/mL) (Table  3 ), this observation has not yet been confirmed using in animal studies.

Conclusions

Our results provide further scientific evidence supporting the use of P. africanum, Z. capense and C. anisata as anti-inflammatory and pain relief remedies in traditional medicine. To be used as herbal products the safety in animal experiments have to be confirmed. The good inhibitory activity of crude extracts containing many other compounds on 15-LOX inhibition in these plant species means that it probably contains compounds with excellent activities. Further work is required to isolate, identify and characterize the bioactive compounds that are responsible for the activities. Once the active compounds have been isolated the mechanism of activity can be examined.

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Acknowledgement

The Tshwane University of Technology, Pretoria, South Africa, and the National Research Foundation (South-Africa) provided funding. This study was carried out at in the Phytomedicine Programme of the University of Pretoria. Mr. Jason Sampson, the curator of the Manie van der Schijff Botanical Gardens, University of Pretoria provided support in plant collection and identification.

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The biological assays analyses and writing the draft manuscript were done by SAA and JPD. LJS critically reviewed the manuscript and participated in the study design and choice of assay methods. JNE conceived the idea, reviewed the draft and final manuscripts and interpretation of results. All authors read and approved of the final manuscript for submission.

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Adebayo, S.A., Dzoyem, J.P., Shai, L.J. et al. The anti-inflammatory and antioxidant activity of 25 plant species used traditionally to treat pain in southern African. BMC Complement Altern Med 15 , 159 (2015). https://doi.org/10.1186/s12906-015-0669-5

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• Anti-inflammatory
• Medicinal plants
• 15-lipoxygenase
• Nitric oxide
• Peltophorum africanum

BMC Complementary Medicine and Therapies

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In vivo anti-inflammatory, antipyretic, analgesic activity and in vitro anti-proliferative activity of aqueous methanolic extract of Euphorbia granulata Forssk

• Mohsin Ahmad Ghauri 1 ,
• Liaqat Iqbal 1 ,
• Ali Raza 2 ,
• Uzma Hayat 2 ,
• Naveel Atif 3 &
• Aqeel Javeed   ORCID: orcid.org/0000-0002-7452-7185 1  

Future Journal of Pharmaceutical Sciences volume  7 , Article number:  34 ( 2021 ) Cite this article

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Naturally occurring substances of plant origin have long been used in folk medicine for curing various ailments including fever, pain, and inflammation etc. After careful evaluation on scientific bases, a large number of those substances provides cheaper alternative to currently used synthetic or semi-synthetic agents. Thus, with an aim of discovering alternative medicine for treatment of such ailments, current study was carried out. Euphorbia granulata Forssk. had long been used as a therapeutic agent against various morbid conditions, e.g., anthelmintic, snake bite, scorpion sting, purgative, and diuretic, and as blood purifying agent in folk medicine. The purpose of the current study was to determine the extended therapeutic use of Euphorbia granulata Forssk. based upon scientific evaluation, to explore the potential of its anti-proliferative, analgesic, antipyretic, and anti-inflammatory activities while using an aqueous methanol extract of the whole plant.

In vivo study was performed on female rats of specie Rattus norvegicus weighing (100–150 g). Anti-inflammatory activity of the plant extract was calculated against using carrageenan induced paw edema. Analgesic potential both central and peripheral was assessed by using Eddy’s hot plate method and acetic acid-induced writhing model, respectively. The antipyretic potential was appraised using brewer’s yeast suspension, injected under the nape of the neck, and body temperature was measured using a digital thermometer. The plant extract strengths used for in vivo experiments were 50 mg, 100 mg, and 200 mg/kg (diluted in normal saline) and were administered through intra-peritoneal route. MTT assay was performed to estimate in vitro anti-proliferative potential. For this assay, a serial dilution of the plant extract was used with 100 μg/ml as the highest concentration. In vivo results demonstrated that plant extract at dose strength of 200 mg/kg, showed significant ( p * < 0.05) anti-inflammatory, analgesic, and antipyretic activities. In case of MTT assay, however, no significant anti-proliferative activity ( p > 0.05) was observed up to 100 μg/ml dose strength.

It can be concluded that aqueous methanol extract of Euphorbia granulata (whole plant) have shown significant anti-inflammatory, analgesic, and anti-pyretic activity in animal model. Therefore it can be a potential candidate, as a therapeutic alternative against treatment of algesia, pyrexia, and inflammation of various pathological origin. However, the plant extract did not demonstrate any significant anti-proliferation activity at doses used in this study.

The naturally occurring plants are an excellent source of various therapeutic substances. The obtained active ingredients from these plants were used as remedy to combat various ailments [ 1 ]. Euphorbia granulata Forssk. (EG) have its place in the plant family Euphorbiaceae. It is locally found in African countries, distributed from drier ranges from Western Sahara and Morocco to Somalia, different countries of Middle East to Kenya and Tanzania, and from Arabian Peninsula and central Asia to China, Pakistan, and India [ 2 ]. Euphorbiaceae is among the family of flowering plants, comprising five subfamilies and approximately 300 plus genera and more than 7000 species [ 3 ]. EG is densely growing branched, annual, or perennial herb [ 4 ]. The plant extract is used as an anthelmintic agent, the latex is topically applied to treat scorpion stings and snake bites. It is also used as a blood purifier, diuretic agent, and purgative [ 5 ]. The plant has also demonstrated HIV-1 and HCV protease inhibitory potential [ 6 ]. The inflammation is believed to be a significant physiological defense mechanism that supports the body to pledge itself against allergens, toxic chemicals, blisters, infection, and various other noxious stimuli [ 7 ]. Uncontrolled and obstinate process of inflammation could be an etiologic factor for some of these enduring ailments. Even though the process of inflammation is a body’s safeguard mechanism, yet the complicated events and mediators involved in the inflammatory process can be easily prompted [ 8 ]. Several natural substances of plant origin exhibit significant anti-proliferative propensity, e.g., Vinca alkaloids from periwinkle plant Catharanthus roseus . A large number of substances of plant origin are already under investigation for their anti-proliferative properties [ 9 ]. Analgesic is any substance that is used to provide relief from pain. The word analgesia originated from Greek and comprises of two parts, “an” meaning without and “gesic” that means pain. So the scholarly sense of analgesia is without pain. These representatives are commonly recognized as pain relievers. These drugs can act on peripheral and central nervous system through various mechanisms. These agents act differently from anesthetics to allay pain (reversibly eliminate agonizing sensation) [ 10 ]. The word antipyretic is also derived from two Greek words, “anti” means against and “pyretic” means fever. Antipyretic hence refer to those agents that reduces fever. These agents guide the hypothalamus brain region to overcome an interleukin-induced upsurge in temperature [ 11 ]. The body then acts through discharge of diverse substances to drop the temperature and diminish the fever [ 12 ]. The undesirable effects escorted with currently available synthetic anti-inflammatory, analgesic, anti-proliferators, and antipyretic drugs pose a major problem during their clinical use.

In the current study, the aqueous methanol extract (70:30) of the EG whole plant was utilized to appraise its analgesic, anti-inflammatory, and antipyretic potential in animal model, whereas MTT assay was performed to determine in vitro anti-proliferative potential of EG in human breast cancer cell lines MCF-7, MDA-MB-231, and SkBr3. Female Wistar rats of specie Rattus norvegicus were used for the in vivo study, the animal selection was based upon other activities of under-observation plant in different literature with same strain of experimental rats [ 13 , 14 , 15 , 16 , 17 ]. To the best of our knowledge, no work has yet been carried out to investigate the in vitro anti-proliferative and in vivo anti-inflammatory, analgesic, and antipyretic effect of whole plant of EG. Therefore, with a view to discover alternative therapeutic options, with minimum side effects for aforesaid ailments, the current study was carried out.

Plant material

EG is a prostrate (stretched out with face on the ground in adoration), annual plant with branches up to 15 cm long; the whole plant appearance is short-hairy or sparsely furry. The plant is typically collected from the wild for native use. The official name of the plant is Euphorbia granulata Forssk. Common names include Euphorbia forsskaolii, Euphorbia turcomanica, lubaina, and spurge. Its local name is lubaina. In English, it is called as desert spurge. Whole plant EG was collected during the month of July–August (2017) from the peripheral areas of Khanewal city in Punjab province, Pakistan. Botanical identification was done from Government College University, Lahore, and a sample was retained there in the herbarium with a voucher number 3820/Bot. Moreover, the plant name has been checked with http://www.theplantlist.org for the accepted name in accordance with the International Plant Names Index (IPNI).

Preparation of plant extract

The non-essential elements from the collected material were detached mechanically and the whole plant was washed three times using distilled water. Subsequently, plant material was shade dried at temperature ranging between (21–30 °C) for 30 days. The dehydrated plant material was mechanically condensed to granular powder and stored in an air-tight vessel. From the obtained plant powder, approximately 500 g of material was taken in a large beaker thereafter; 2 liters of dichloromethane (DCM) was then added to it, and the mixture was then macerated for 72 h and then again macerated with methanol for 72 h with intermittent shaking and stirring. The filtrate was extracted three times with the fresh solvent and all the extract was then combined. Multi-layered muslin cloth was used for coarse filtration of the macerated plant material. Once the coarse filtration is completed, the plant extract was filtered through Whatman no. 1 filter paper for fine filtration. The acquired filtrate was then resolved under reduced pressure (760 mmHg) at 40 °C in a rotary evaporator (Heidolph Laborota 4000, USA). The semisolid mass assimilates indicated percentage yield of 29.6%, and later it was desiccated in an oven at 40 °C. To speed up the process of extraction, grinding was done and later size reduction was performed with a pestle and mortar. After size reduction, plant material was macerated in (70:30) water and methanol. The grinded material was macerated in aqueous methanol mixture three times, then filtered and evaporated on rotary evaporator [ 18 , 19 ].

Solvents and chemicals

The standard drug indomethacin was locally acquired from Chiesi Pharmaceuticals, Pakistan; tramadol (Tonoflex injection) and paracetamol (Provas injection) were purchased from SAMI Pharmaceuticals Pvt. Ltd. Acetic acid and brewer’s yeast was purchased from Punjab Chemicals Pvt. Ltd. Lahore, Pakistan. Carrageenan was purchased from Sigma Aldrich, USA. Sterile normal saline (NS) (PAKSOL pharmaceuticals pvt Ltd) and sterile water for injection was purchased from local distributer, Muller and Phipps (M&P), Lahore, Pakistan. Dimethyl sulfoxide (DMSO) from Sigma Aldrich, USA; Dulbecco’s modified Eagle medium (DMEM); Roswell Park Memorial Institute (RPMI 1640) and L15 complete medium from (Hyclone, USA); penicillin/streptomycin solution (Hyclone, USA); phosphate buffer saline (PBS; Hyclone, USA); fetal bovine serum (FBS; Hyclone, USA); MTT reagent (Bio world, Dublin); and trypsin (Hyclone, USA). The doses of the plant extract were prepared in DMSO and diluted in sterile normal saline under aseptic laboratory conditions in a clean room for all the experiments.

Experimental animals

With prior approval from the ethical committee for the animal experiments (Institutional Review Committee for Biomedical Research, University of Veterinary and Animal Sciences, Lahore, Pakistan), approval number is IRCBR/886-E-17/PCOL/UVAS. Wistar rats (female) aging 1 to 2 months of Rattus norvegicus species weighing 100–150 g were purchased from University of Health Sciences (UHS), Lahore. The experimental rats, (ER), now onwards, were retained under routine laboratory conditions of 22–25 °C with alternate light/dark 12/12-h period. Pellet form feed was given to ER and water ad libitum. For every experiment, 20 ER were used and distributed randomly in five groups and each group comprises of four ER.

Anti-inflammatory activity

The anti-inflammatory strength of aqueous methanolic extract of whole plant was assessed against using carrageenan-induced paw edema model. ER were randomly divided into five groups counting four ER in each group. ER were deprived of feed 1 h before experiment. Group I was administered sterile normal saline as blank or negative control 10 ml/kg; group II was given standard drug indomethacin 10 mg/kg, as positive control; group III, IV, and V were given plant extract 50, 100, and 200 mg/kg, respectively. After 1 h of intra-peritoneal (i.p.) administration of standard drug and plant extract, 1% carrageenan solution approximately 100 μl was injected into the left hind paw of each ER as an inflammatory mediator. The paw volume of each ER was then determined immediately at 0 h and after 3 h of carrageenan injection using liquid immersion model [ 20 ]. To measure the volume of the paw edema, a glass beaker was filled with distilled water and positioned on a weighing balance; the weight of the beaker was tared. The paw volume of each ER was measured by immersion of the inflamed paw into water. A force F is applied to the balance against the movement of liquid inside the beaker and that was equal to weight of the paw. The paw volume was measured using Eq. ( 1 ).

As the specific gravity of water at room temperature is taken as 1 so each 1 g increase in weight is equal to the 1 cm 3 increase in volume.

The mean paw edema was calculated for all groups and compared, with that of negative control group. Percentage inhibition of the inflammation was then measured using Eq. ( 2 ) given as:

where Vt is paw volume at time t = 3 h and Vo is volume at 0 h time.

Analgesic activity

Eddy’s hot plate model.

The ER were randomly distributed into five groups comprising four ER in each group. All animals were introverted from feed 2 h before the beginning of experiment. Pre-testing of ER on Eddy’s hot plate kept at 55 °C ± 0.1 °C was then performed. ER displaying latency time greater than 15 s were omitted from the experiment. In our study, none of the ER displayed latency time greater than 15 s in pre-testing examination so there was no exclusion. The groups were injected with the following: group I was injected sterile NS 10 ml/kg; group II was given tramadol 20 mg/kg; group III, IV, and V were given EG 50, 100, and 200 mg/kg via i.p. route of administration, respectively. Thirty minutes after administration of corresponding treatment, the ER were placed on Eddy’s hot plate and latency time (time for which ER remained on the hot plate without licking or flicking of hind limb or jumping) was then measured for 1 min and recorded [ 21 ]. The percentage analgesia was calculated by Eq. ( 3 ).

Writhing model

ER were divided into five groups comprising four ER in each group. The peripheral analgesic potential was assessed by acetic acid-induced abdominal writhing. The groups were given with following: group I was administered sterile NS 10 ml/kg; group II was administered indomethacin 10 mg/kg; group III, IV, and V were administered plant extract 50, 100, and 200 mg/kg, respectively. After group treatment, approximately 50 μl of 1% acetic acid solution in sterile NS was given to each ER through i.p. route of administration. Twenty minutes later, the abdominal constrictions or writhing was observed and counted for 1 min and recorded [ 22 ].

Antipyretic activity

Brewer’s yeast-induced pyrexia model.

Antipyretic strength of the plant extract was appraised against using methods previously described. ER were randomly allocated into five groups comprising four ER in each group. The standard body temperatures of the ER were examined by inserting a digital thermometer into their anal cavities for about 1 min. The rectal temperature readings observed were recorded as pre-treatment or normal body temperature. Thereafter, pathogenic fever was induced in ER by injecting 15% brewer’s yeast ( Saccharomyces cerevisiae ) suspension at dose adjustment of 1 ml/kg, subcutaneously under the nape of the neck and 24 h later the rectal temperature of all the ER was measured again. ER that did not show a baseline increase of 0.3 °C temperature was excluded from the study. In our study, none of the ER was omitted due to the aforesaid criteria. Later on, group I was given sterile NS 10 ml/kg; group II received standard drug paracetamol 150 mg/kg; and group III, IV, and V were given plant extract 50, 100, and 200 mg/kg, via i.p. route of administration, respectively. Rectal temperature was then noted directly at 0 h and after 1, 2, 3, and 4 h post drug treatment [ 23 , 24 ].

Sterility test

Preparations were intended to be injected through intra-peritoneal route of administration, so they needed to comply with sterility protocol. Following the standards of the International Pharmacopoeia, we cross-checked by carrying out sterility test using fluid thioglycolate medium for anaerobic microbes and soya bean-casein medium for aerobic microbes, incubated for 14 days with preparations separately under the same conditions in which original tests were performed. The intended preparations were inoculated with equal volumes of mentioned medium. Thereafter, we have checked the appearance of any visible growth of microbes at 3rd, 5th, 7th, 9th, 11th, and 14th day after inoculation. In macroscopic examination, no visible growth or turbidity were observed in any of the culture plate. Hence, it was ascertained that plant extract and standard drugs used earlier were all sterile by replicating the similar conditions as were used during experiments [ 23 ].

In vitro anti-proliferation test

Cell culture.

Human breast cancer cell lines MCF-7, Skbr3, and MDA-MB-231 were provided by the Centre of Excellence in Molecular Biology (CEMB), PU Lahore. Receptor-based classification categorizes these cells into estrogen positive (ER+), e.g., MCF-7, Human epidermal growth factor receptor-2 positive (HER2), e.g., Skbr3 and triple-negative breast cancer (TNBC) type cancer cells, which do not possess any of the classical receptors of breast cells, e.g., MDA-MB-231 cell line. The MCF-7 cells were cultured in DMEM and Skbr3 cells were cultured in RPMI 1640, whereas MDA-MB-231 cells were grown in L15 complete medium; all the medium were supplemented with 10% FBS (v/v) and 1% penicillin-streptomycin solution (v/v) at 37 °C, 5%CO 2 , and 95% relative humidity except for MDA-MB-231 cell line, which was incubated in CO 2 -free environment.

The cytotoxic potential of EG whole plant extract was appraised as described before with slight modifications. Briefly, the cells were seeded into 96-well plate at a density of 2 × 10 4 cells/well and incubated for 24 h, followed by the treatment with different dilutions of plant extract (0.78,1.56,3.12, 6.25,12.5,25,50,100 µg/ml) for 48 h. After 48 h, MTT reagent (5 mg/ml) was added in each well with serum-free medium (200 μl) and subjected to further incubation for 4–6 h; thereafter, the contents were removed from each well. In order to dissolve the formed crystals of purple formazan, 150 μl of DMSO was added to each well and kept for 20 min at 37 °C on an orbital shaker for 15 min. The absorbance was then calculated using an ELISA plate reader at 490 nm. Absorbance of control (without any treatment) was used as reference for calculation of cytotoxicity and cell viability [ 25 ].

Statistical analysis

All the data were expressed in terms of mean ± S.D. The experimental results were analyzed for statistical significance by one-way ANOVA followed by post hoc Dunnett’s test for multiple comparisons. p < 0.05* was considered significant.

In carrageenan-induced paw edema, the plant extract demonstrated dose-dependent anti-inflammatory activity. The maximum anti-inflammatory activity (59.12%) of the plant extract was noticed at a dose level of 200 mg/kg as compared to the reference drug indomethacin 10 mg/kg with 81.97% of edema inhibition (Fig. 1 ).

a Anti-inflammatory effects demonstrated as paw volume and b as percentage inhibition 3 h after group treatment of EG ( n = 4). Asterisk shows significant difference ( p < 0.05) between treated group and control

The study demonstrated that the plant extracts EG revealed substantial peripheral as well as central analgesic trait, at a dose strength of 200 mg/kg. The central analgesic property was evaluated and the plant extract of EG displayed dose-dependent increase in latency time of lifting the paw on Eddy’s hot plate and the analgesic potential was found to be 61% at 200 mg/kg dose as compared to 86% for reference drug tramadol 20 mg/kg. The results for latency time were recorded at 0, 20, and 60 min (Fig. 2 ).

a Analgesic property demonstrated as latency time after different time intervals of dose administration and b percentage analgesia of plant extract ( n = 4). Asterisk shows significant difference ( p < 0.05) between treated group and control

The peripheral analgesic activity was estimated by acetic acid-induced writhing model and the number of writhings were counted for 60 s and 20 min after injection of 1% acetic acid to all groups. The difference between the number of writhing’s in the control group and treatment groups was then calculated and percentage analgesia was determined. Plant extract produces a 27% decrease at 50 mg/kg, 41% at 100 mg/kg, and 46% at 200 mg/kg dose in writhing response when compared with the control group. The maximum analgesic strength of the plant extract for peripheral analgesic activity was found to be 46% at a dose strength of 200 mg/kg as compared to 64% for indomethacin taken as standard (Fig. 3 ).

a Peripheral analgesic potential demonstrated by number of writings and b as percentage analgesia of plant extract for 20 min after i.p. administration of given doses of EG aqueous methanolic extract at 50, 100, and 200 mg/kg doses ( n = 4). Asterisk shows significant difference ( p < 0.05) between treated group and control

Antipyretic activity of aqueous methanolic extract of plant was calculated by first inducing pyrexia to experimental animals and then injecting various concentration of plant extract and compared against established reference drug and negative control groups. The plant extract at 200 mg/kg showed significant antipyretic potential against brewer’s yeast-induced pyrexia in animal model. The rectal temperature of ER was recorded immediately at 0 h, then after 1, 2, 3, and 4 h, after the plant extract treatment. The results compiled thus showed that aqueous methanolic extract of EG whole plant demonstrates significant antipyretic activity at dose of 200 mg/kg comparable to the reference drug paracetamol used at a dose of 150 mg/kg (Fig. 4 ).

Body temperature after treatment of pyrexia in ER ( n = 4). Asterisk displays significant variance ( p < 0.05) between treated groups reference and negative control group

Anti-proliferation activity

The results of MTT assay using aqueous methanolic extract of whole plant EG are shown in Fig. 5 . The findings of MTT assay indicated that the under-observation plant extract at given doses did not show any significant anti-proliferation activity in breast cancer cell lines MCF-7, Skbr3, and MDA-MB-231 (Fig. 5 ).

Cell viability and dose inhibition curves for breast cancer cells (MCF-7, Skbr3, MDA-MB-231). a Cell viability of receptor-based classified breast cancer cell lines incubated for 48 h with different concentrations of EG-CR extract. b Dose inhibitory curve in breast cancer cell lines against log of different concentration of EG extract ( n = 4)

Therapeutically valued substances of plant origin were used as a source of treatment strategy against various pathological conditions in ancient days. In folk medicine, these plants derived substances were used for many years in curing of various ailments including fever, pain, and inflammation [ 26 ]. The plant EG is one of the better reported plants with several therapeutic applications. It was chosen to evaluate and validate the scientific basis of its traditional use for the aforesaid conditions [ 27 ]. In the current study, the plant EG was evaluated for its anti-proliferative, anti-inflammatory, analgesic, and antipyretic potential. Aqueous methanolic extracts of whole plant of EG was prepared according to methods previously described [ 28 ]. Carrageenan-induced inflamed paw model was used to evaluate anti-inflammatory activity of EG extract. The release of certain inflammatory and pro-inflammatory substances, e.g., histamine, bradykinin, prostaglandins, and leukotrienes, are influenced by carrageenan [ 29 ]. Indomethacin was taken as standard control for the anti-inflammatory test of plant extract. The onset of paw edema prompted by carrageenan is a biphasic event. In the first phase of episode, the discharge of numerous inflammatory mediators occurs, e.g., kinins, histamine, and serotonin, while the second phase is primarily connected with the discharge of bradykinins and prostaglandins [ 30 ]. The size of the paw was taken as the parameter, to quantify inflammation and resultant increase in size is directly proportional to edema. The outcome of the statistical analysis has shown that plant extract at dose strength of 200 mg/kg produced a significant ( p < 0.05) decrease in edema after 3 h, of treatment. The plant extract exhibits graded dose response relationship against edema and it was noted that at 200 mg/kg dose, it produced 59% decrease in edema. The acute inflammatory model used in research studies is generally established by carrageenan chemical that is subtle to cyclooxygenase inhibitors [ 31 ]. This model is most frequently used to find out the result of NSAID’s which favorably detain cyclooxygenase enzyme involved in prostaglandin synthesis [ 32 ]. Although lipoxygenases and cyclooxygenase both pathways are connected with facilitating inflammatory development but inhibitors of the cyclooxygenase rather than lipoxygenases pathway are assumed to be more useful [ 33 ]. The process of inflammation is coupled with an increase in number of prostaglandins, leukocytes, and numerous other inflammatory mediators at the specific site [ 34 ]. The anti-inflammatory properties of plant extract EG consequently related with the inhibition of cyclooxygenase enzyme which in turn stops prostaglandin synthesis [ 35 , 36 ]. In addition to that, flavonoids are the chemicals which constrain prostaglandin synthetase enzyme activity, so the estimation of anti-inflammatory potential also confirms the existence of flavonoids in the plant extract [ 37 ]. Substances involved in attenuating sensations of pain are termed analgesic. These agents are way different from anesthetics, because the later induce a reversible or temporary lessening in pain perception. The writhing movements are instigated by the peritoneal receptors that exist in the abdominal region. Discharge of various endogenous substances, e.g., arachidonic acid is the cause of pain initiation [ 38 ]. In acetic acid-triggered writhing model, the root cause of pain is the native discharge of arachidonic acid and prostaglandins. Administration of acetic acid causes release of certain endogenous substances that excite nerve endings of pain receptors [ 39 ]. Aqueous methanol extract of EG demonstrate significant peripheral analgesic effects that is comparable to that of standard drug indomethacin. The induction of writhing model for evaluation of peripheral analgesic effect is thought to be classier than tail flick model. Local peritoneal receptors are supposed to be involved in abdominal contraction response [ 40 ]. Evidence advocates an increase in level of lipoxygenase and that of PGF2α and PGE2 in peritoneal fluid. It is also believed that there occurs an increase synthesis of nitric oxide. The drug tramadol resembles opioid in its mode of action. Investigational aqueous methanolic plant extract showed dose-dependent increase in central analgesic effects. The mechanism through which the plant extract possibly exerts this effect is the inhibition of prostaglandin biosynthesis [ 41 ]. The algesia is a two-step progression. In the first phase, stimulation of nociceptors occurs, possibly due to the role of bradykinins and substance P. In the second phase, serotonin, histamine, and prostaglandins are produced causing inflammation. Central analgesic agents inhibit both steps of pain progression, whereas peripheral analgesics can only be able to avert the second phase of the pain perception [ 42 ]. In our study, the plant extract exhibited inhibition of both the phases of pain progression, and consequently associated with both central as well as peripheral analgesic qualities. Agents that are able to reduce raised body temperature in certain pathological conditions are generally known as antipyretic substances. In our study, administration of brewer’s yeast suspension to the experimental animals results in increase in prostaglandins synthesis and onset of pyrexia [ 43 ]. It is a valuable model for evaluating antipyretic activity not only for the plant-derived substances but also for the synthetically produced chemicals. Pyrexia induction to the animal model by injecting yeast suspension is called pathogenic fever, and it is associated with increased prostaglandin production [ 44 ]. The plant extract constrains cyclooxygenase enzyme subsequently decreases the discharge of prostaglandins as a result poses its antipyretic properties. The i.p. administration of plant extract has displayed substantial antipyretic trait in the brewer’s yeast elicited pyrexia in animal model. Anti-proliferative potential of the plant extract was evaluated against using cell proliferation assay previously described with little modifications [ 45 ]. MTT assay is the most common way to ascertain anti-proliferative potential of the various substances, using different dilutions. In our study, however, EG extract showed no significant anti-proliferative activity against receptor-based classified different types of breast cancer cell lines. Moreover, further purification of the plant extract will be required to optimize the therapeutically acceptable dose range as well as the toxicity studies, for maximizing the best possible therapeutic window, that may be used in the future as an alternative treatment strategy. In addition to that, dose adjustment with maximum tolerability to the normal cells and maximum sensitivity to tumor cells may be applied to different tumor models, so as to ascertain potential anti-proliferation activity of the under observation EG plant extract.

Overall findings of our study revealed that EG aqueous methanol extract has got significant anti-inflammatory, antipyretic, and analgesic properties at dose strength of 200 mg/kg. However, no significant in vitro, anti-proliferative activity of plant extract was observed with various dose-dilutions. Moreover, in the future, further studies will be required, as far as extended therapeutic use of under-observation plant is concerned, to advocate its dose related anti-proliferation property, within tolerable range to the growth of normal cells and at the same time toxic to cancer cells, in various cancer models.

Availability of data and materials

Data and material are available upon request.

Abbreviations

• Euphorbia granulata

Experimental rats

Normal saline

Intra-peritoneal

Dimethyl sulfoxide

Dulbecco’s modified eagle medium

Roswell Park Memorial Institute

Phosphate buffer saline

International Plant Names Index

(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

Enzyme-linked immunosorbent assay

Non-steroidal anti-inflammatory drugs

Dichloromethane

Fetal bovine serum

Carbon dioxide

Centre of Excellence in Molecular Biology

Estrogen receptor positive

Human epidermal growth factor receptor-2

Triple-negative breast cancer

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Acknowledgements

We are thankful to the Institute of Pharmaceutical Sciences, University of Veterinary and Animal Sciences, Lahore, Pakistan, for providing facilities to conduct part of our research work. We are highly thankful to all laboratory staff of pharmacology department, who were always there for assistance during the University off hours as well.

Plant authentication

Botanical identification of under-observation plant Euphorbia granulata Forssk. was done from Government College University Lahore, and a sample was retained there in the herbarium with a voucher number 3820/Bot. Moreover, the plant name has also been checked with http://www.theplantlist.org for the accepted name in accordance with the International Plant Names Index (IPNI).

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Authors and affiliations.

Department of Pharmacology and Toxicology, University of Veterinary and Animal Sciences, Lahore, Pakistan

Mohsin Ahmad Ghauri, Liaqat Iqbal & Aqeel Javeed

University College of Pharmacy, University of the Punjab, Lahore, Pakistan

Ali Raza & Uzma Hayat

Center of Drug Safety and Policy Research, School of Pharmacy, Xi’an Jiao Tong University, Xi’an, Shaanxi, China

Naveel Atif

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MAG and LI designed all experiments, performed experiments, and wrote manuscript. AR analyzed the data. UH and NA technical assistance and support. AJ supervised the work and provided reagents and information. The authors read and approved the final manuscript.

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Animal experimentation were performed after the approval from University animal experiment ethical committee (Institutional Review Committee for Biomedical Research University of Veterinary and Animal Sciences Lahore, Pakistan) IRCBR/886-E-17/PCOL/UVAS, as no toxicity study was involved in our research work; the rats were given to the animal house of the university in good health condition.

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1. Anti-inflammatory activity. 2. Analgesic activity (central). 3. Analgesic activity (central). 4. Analgesic activity (central). 5. Anti-Proliferative activity.

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Ghauri, M.A., Iqbal, L., Raza, A. et al. In vivo anti-inflammatory, antipyretic, analgesic activity and in vitro anti-proliferative activity of aqueous methanolic extract of Euphorbia granulata Forssk. Futur J Pharm Sci 7 , 34 (2021). https://doi.org/10.1186/s43094-021-00184-9

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Received : 21 February 2020

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Published : 04 February 2021

DOI : https://doi.org/10.1186/s43094-021-00184-9

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Anti-Inflammatory and Antioxidant Properties of Plant Extracts

María jesús rodríguez-yoldi.

1 Department of Pharmacology and Physiology, Veterinary Faculty, University of Zaragoza, 50013 Zaragoza, Spain

2 CIBERobn, ISCIII, IIS Aragón, IA2, 50009 Zaragoza, Spain; se.razinu@loydorjm

Since the ancient times, a great variety of plants have been used for therapeutic purposes. Most parts of plants have been used as extracts and may possess anti-inflammatory and antioxidant properties related to diseases such as diabetes, atherosclerosis, neurodegenerative, or cancer. In addition, plant extracts, as anti-inflammatory agents, can regulate the composition of the gut microbiota.

Fruits and vegetables contain a large amount of compound phytochemicals responsible for their medicinal properties, such as polyphenols, carotenoids, phytosterols, and polysaccharides. Currently, phytochemical and ethnobotanical studies are being carried out in order to identify the mechanism of action of a wide variety of natural compounds present in plant extracts.

In this way, certain ailments whose etiology involves immune dysfunction or persistent inflammation can be protected by plants consumption by the downregulation of pro-inflammatory cytokines, COX, and reducing the translocation of NF-kB to the nucleus. Also, bioactive principles of plants can regulate the oxidative stress caused from an imbalance in the production of reactive oxygen species (ROS) and the antioxidant capability of the cell enzymes.

This Special Issue of Antioxidants is dedicated to current research on the aspects related to the anti-inflammatory and antioxidant properties of plant extracts, as well as the modulators and pathways involved in these therapeutic actions. In this issue, 16 original research papers and 6 reviews have been published.

In this context, it is known that polyphenols are the most abundant antioxidants in the human diet. They represent a variety of bioactive compounds that are capable of preventing and controlling diseases related to stress diseases. Thus, the review realized by Jung et al. provides an overview of the therapeutic benefits and targets of Salvia miltiorrhiza extracts, including inflammation, fibrosis, oxidative stress, and apoptosis [ 1 ]. However, it must be taken into account that the content of polyphenols in plants varies remarkably between species, geographical area, part of the plant tested, etc. In this sense, Bhatt and Debnath [ 2 ] studied, in blueberries ( Vaccinium spp.), the genetic diversity of antioxidant properties in relation to the total phenolic and flavonoid content, and associated with molecular markers.

Phytosterols belong to the triterpene family and are structurally similar to cholesterol. They are known for their cholesterol-lowering effects, anti-inflammatory and antioxidant properties, and the benefits they offer to the immune system. The review by Vezza et al. provides an overview of these bioactive compounds and their therapeutic potential in the fields of obesity and metabolic disorders, in relation to oxidative stress, inflammatory status, and gut dysbiosis [ 3 ].

As indicated, plant extracts have been used as natural therapeutic agents against inflammation, characterized by an overproduction of inflammatory mediators such as ROS and pro-inflammatory cytokines. In this sense, studies in murine macrophage cells (RAW264.7), with extracts from Notopterygium incisum, revealed an anti-inflammatory effect by interfering with lipopolysaccharide (LPS) binding to Toll-like receptor 4 (TLR4), signaling and activating the antioxidative nuclear factor erythroid 2-related factor 2 (Nrt2). In addition, this factor increased the expression of the antioxidant protein heme oxygenase-1 (HO-1), superoxidase dismutase (SOD) and catalase (CAT) [ 4 ]. Similar results were obtained with different organ extracts of Abeliophyllum distichum by suppressing ROS production and/or inhibiting the activation of protein kinases (ERK1/2) when treating RAW264.7 cells with LPS [ 5 ]. Likewise, in LPS-activated RAW2647.7 cells, sorghum extracts inhibited the production of nitric oxide (NO), interleukin-6 (IL-6), and ROS, with ethanolic extracts showing greater anti-inflammatory activity related to tannin content [ 6 ]. Berries are a rich source of phytochemicals, especially phenolics, which are well known for their protective activity against many chronic diseases. In addition, berries also contain a complex mixture of volatile compounds that are responsible for its aroma. Gu et al. [ 7 ] found that both berry phenols and volatile fractions show an anti-inflammatory effect on LPS-stimulated RAW264.7 cells through the suppression of the nuclear factor-kappa B (NF-kB) signaling pathway.

The inflammation of the colon is a highly currently prevalent disease. Kopiasz et al. found that oat beta-glucans exert an antioxidant effect in animals with colitis induced by 2, 4, 6-trinitrobenzene sulfonic acid (TNBS), with greater effectiveness in removing the systemic effects of colon inflammation [ 8 ].

Increased levels of ROS and a low-grade chronic inflammation in multiple organs have been demonstrated in obesity. A comparative study of the antioxidant and anti-inflammatory effects of leaf extracts from four different Morus alba L. genotypes (Filipina, Valenciana Temprana, Kokuso, and Italia), in mice with diet-induced obesity, was performed by Leyva-Jimenez et al. [ 9 ]. The results showed that Filipina and Italia methanolic extracts have the higher antioxidant and anti-inflammatory effect due to the presence of compounds such as protocatechuic acid or quercetin-3-glucoside, and they could be developed as a complementary treatment for obesity and metabolic disorders. Continuing with studies related to inflammation diseases, Mainka et al. determined the antioxidant activity and composition of extracts from eleven species of plants traditionally used in inflammatory skin diseases in Poland [ 10 ].

Likewise, a review by Noh et al. showed that several natural products possess antioxidant properties and androgenic activities on productive factors and hormones, and therefore the management of infertility with these products could be taken into account [ 11 ].

Regarding to the powerful antioxidant activity of polyphenols, these compounds can help prevent or treat several diseases related to oxidative stress such as cancer, diabetes, neurodegenerative, autoimmune, cardiovascular, and ophthalmic diseases, among others [ 12 ]. However, plant extracts can also be used to alleviate the harmful side effects of drugs. Thus, a study by Abd El-Rahman et al. [ 13 ] showed that Saussurea lappa has a remarkable protective effect against triamcinolone acetonide (glucocorticoid extensively used) via its anti-inflammatory, anti-apoptotic, and antioxidant capacity. In addition, the plant extract can be a viable alternative to the serious problem of microbial resistance to antibiotics. In this way, natural extracts from Pinus pinaster bark, rich in phenols, showed potent antibacterial activity against Gram-positive bacteria [ 14 ]. Likewise, a review by Bogdan et al. shows the antimicrobial activity of Vitis vinifera by-products and their application in oral care [ 15 ].

The antioxidant capacity of polyphenols can also be used as an anti-aging potential, as is shown in a study carried out by Moliner et al. [ 16 ] with rosemary flowers ( Rosmarinus officinalis L.), in Caenorhabditis elegams .

A large number of studies have suggested that the consumption of polyphenols could have beneficial effects on the central nervous system, by improving its blood flow as well as preventing or delaying the onset of neurodegenerative disorders [ 12 ]. In this way, peripheral neuropathy (PN) is a clinical problem that affects many patients and with few effective therapies. Recently, the interest in natural dietary compounds in human health has led to a great deal of research, especially in PN. Curcumin is a polyphenol extracted from the root of Curcuma longa , and has been used in Asian medicine for its anti-inflammatory, antibacterial, and antioxidant properties. In a review by Caillaud et al., in vivo and in vitro studies on this molecule in the treatment of different PN have been shown, highlighting its molecular mechanisms of action [ 17 ].

Dietary intervention could play a key role in both the prevention and treatment of diabetes mellitus. Pancreatic beta cells are vulnerable to oxidative stress, which causes beta cell death and dysfunction in diabetes mellitus. In this sense, Broussonetia kazinoki Siebold was used to prevent or treat beta cell damage in diabetes [ 18 ]. Likewise, Pinus pinaster bark extracts showed hyperglycemic activity determined by α-amylase and α-glucosidase assays [ 14 ].

Cancer is one of the leading causes of death in the world. Cancer treatments, based on chemotherapy, surgery, radiotherapy, etc., are currently applied. However, sequela of such cancer therapies and cachexia are a problem to the patients, hence the interest in applying natural compounds in the treatment of this disease. A review by Lee et al. focuses on the potential of plant extracts as great therapeutic agents in controlling oxidative stress and inflammation associated with tumoral environment. Thus, this review shows how some antioxidants derived from plants inhibited the proliferation of cancer cells and inflammation after surgery, and others prevented the apoptosis of healthy cells through the elevation of antioxidant enzymes or anti-apoptotic proteins and controlling cytokine levels [ 19 ]. Therefore, dietary polyphenols might play a dual role in cancer treatment, since they have been proved to be beneficial for chemoprevention as well as for cancer treatment. Thus, grape stem extracts showed an antiproliferative effect in colon (Caco-2) and breast (MCF-7 and MDA-MB-231) cell lines through apoptosis cell death associated with a modification of the mitochondrial potential and ROS levels. Additionally, grape stem extracts showed an antioxidant effect on non-cancerous intestinal cells that could protect the intestine from diseases related to oxidative stress [ 20 ]. Likewise, Capsicum annuum L., incorporated into liposomes, reduced the ROS in the human hepatoma (HepG2) cell line, and the extracts promoted the expression of endogenous antioxidants, such as catalase, superoxide dismutase, and glutathione peroxidase through the Nrf-2 pathway [ 21 ]. Pinus pinaster bark extracts also showed an anticancer effect by decreasing cell viability in human lung cancer cells [ 14 ]. Studies realized with Satureja hortensis L. herb have shown that the budding phase alcohol extracts of this plant contain the largest amounts of polyphenols, including rutin and rosemary acid, which promote the radical scavenging activity and antioxidant properties with an anticancer effect on tumor skin cells [ 22 ]. In this way, Phoenix dactylifera seed extract can also be used as a skin whitening agent by attenuating melanogenesis in B16F10 cells by downregulating protein kinase A (PKA) signaling pathways [ 23 ].

Taken together, the papers included in this Special Issue illustrate examples of recent advances in the treatment and prevention of disease with plant extracts. In some work it has been shown that fruit and vegetable residues (waste by-products), rich in bioactive compounds, can also be used for therapeutic purposes. In addition, in some papers, the alternative application of natural compounds in the case of resistance to antibiotics, or in harmful side drug effects, has been shown. The general objective was to compile research and review articles that cover the latest trends in the area, and thus benefit researchers and readers in advancing their knowledge on the subject.

This research received no external funding.

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The author declares no conflict of interest.

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