Analgesic and Antipyretic Activities of Ethanolic Extract of Sappan wood (Caesalpinia sappan L.) Leaves
1Department of Pharmaceutical Analysis and Medicinal Chemistry, Faculty of Pharmacy, Universitas Padjadjaran, West Java, Indonesia – 45363.
2Department of Pharmacy, Faculty of Mathematics and Natural Sciences, Al Ghifari University, West Java, Indonesia – 40293.
*Corresponding Author E-mail: nyi.mekar@unpad.ac.id
Sappan wood (Caesalpinia sappan L.) is used as an analgesic and antipyretic by the Indonesian people, empirically. The aim of this study was to determine the analgesic and antipyretic activity of ethanolic extract of sapan wood leaves in Webster mice as experimental animals. The writhing method was used to determine the analgesic activity in acetic acid-induced mice with mefenamic acid as a positive control. The temperature reduction method was used to determine the antipyretic activity in yeast-induced mice with paracetamol as a positive control. One-way ANOVA was conducted for statistical analysis, followed by Tukey-Kramer post hoc test. Phytochemical screening showed that sappan wood contains alkaloids, flavonoids, saponins, monoterpenoids, and sesquiterpenoids. The optimum dose of analgesic and antipyretic activity was 6.3 mg and
8.4 mg/20 g BW of mice, respectively. The conclusion was ethanolic extract of sappan wood leaves has analgesic and antipyretic activities.
KEYWORDS: Writhing method, Temperature reduction method, Mefenamic acid, Paracetamol.
Sappan wood (Caesalpinia sappan L.) is widely found in India, Malaysia, and Indonesia at 1-700 m in altitude1. This plant belongs to the Caesalpiniaceae family, is traditionally used as an analgesic, antidote, anti- inflammatory, antipyretic, antiseptic, antithrombotic, hemostatic, hepatoprotective, gonorrhea, sedative, and cardiotonic in Indonesia. Empirically, sappan wood is used as a medicine for tuberculosis, rheumatism, dysentery, analgesic, scabies, disinfectant, and tonic2. The taste of sappan wood is sweet, salty, warm, astringent, and odorless3.
Sappan wood contains gallic acid, tannins, phenolic compounds, saponins, pigments, and essential oil which consists of D-a-phellandrene and ocimene4. The phenolic compounds are xanthone, coumarin, chalcones, flavones, flavonoids, and brazilin5.
The world population’s tendency was to use herbal medicines for healthcare needs, due to various ingredients to treat chronic diseases6. This research was the first study that determines the analgesic and antipyretic activity of ethanolic extract of sapan wood leaves (EESL) in Webster mice as experimental animals.
Sappan wood leaves were collected from Sumedang district, West Java, Indonesia, then identified by School of Life Science and Technology, Institute of Technology Bandung, Indonesia with No. 1159/II.CO2.2/PL/2018. Swiss mice (18-25g) were obtained from the Center for Biological Sciences, Institute of Technology Bandung, Indonesia. The mice were fasted overnight, but were provided with water ad libitum before the experiments.
Mefenamic acid and paracetamol with pharmaceutical grade were purchased from China. All chemicals with analytical grade (Merck) were ferric chloride, hydrochloric acid, sodium hydroxide, sodium acetate, n- hexane, methanol, chloroform, sulfuric acid, glacial acetic acid, ethanol, ether, Mayer, Dragendorff, and Bouchardat reagent.
All the experimental procedures and protocols in this study were reviewed and approved by the Research Ethics Committee of Universitas Padjadjaran, West Java, Indonesia with No. 936/UN6.KEP/EC/2018.
Simplicia was extracted with 70% ethanol in a reflux apparatus for 12 h. Each 4 h, the solvent was changed with the fresh one. All extract was collected and evaporated at 40ºC, then calculated the yield7. Phytochemical screening was conducted to simplicia and extract with Farnsworth method8.
The dose of EESL was calculated from the traditional use of sappan wood as an analgesic and antipyretic. Empirically, 9g of sappan wood leaves was boiled with 200mL of water for 15 min9. This dose was converted to mice dose10.
The mice were divided into five groups (n=5), i.e. saline, mefenamic acid (1.3mg/20g BW), and EESL (4.2, 6.3, and 8.4mg/20g BW). Mefenamic acid and EESL were prepared as suspension with 2% arabic gum as a suspending agent. The suspension was administered orally to the mice. After 30 min, mice were induced with 0.7% acetic acid per intraperitoneal. The writhing response was observed form of backward, pulling, retraction, and tetanic spasms by bending the head and legs backward. The observation was recorded every 5 min for 60 min. Percentage inhibition was calculated using the formula 1, with Wc = number of writhing in the control group, Wt = number of writhing in the test group. Compounds with less than 70% inhibition were
considered to have minimal analgesic activity11.
Values were considered statistically significant at p < 0.05.
Sappan wood leaves are Indonesian ethnomedicine. The sappan wood leaves were 10-25mm in length, 3-11mm in width, oval shaped, flat edges, and almost parallel, which accorded with the literature3. Reflux was chosen as the extraction method, the Indonesian people are prepared these herbal medicines by boiling the simplicia. Reflux with solvent replacement was conducted to optimize the secondary metabolites extraction from the simplicia. The water content of sappan wood leaves simplicia was 2%. This value met the requirements, i.e. not more than 10%12.
Phytochemical screening was conducted based on the color alteration method8. Simplicia and EESL contain alkaloids, flavonoids, saponins, monoterpenoids, and sesquiterpenoids. The result of extract phytochemical screening was the same as simplicia, this showed that secondary metabolites were stable while heating. The result of phytochemical screening was in accordance with other study by Kaur et al. on methanolic extract13 and Gunasekaran et al. on ethanolic extract14. A total of 150 g of sappan wood leaves was producing 18.42g of concentrated extract and the yield was 12.28%. The EESL was dark green with a specific odor.
Pain is modulated central mechanism via a complex process, including opiate, dopaminergic, noradrenergic, and serotonergic systems. While peripheral mechanism via prostaglandins, leukotrienes, and other endogenous substances15-17. Acetic acid induces abdominal constriction is a sensitive procedure to evaluate analgesic
18-19
peripherally . Acetic acid-induced writhing causes of
% inhibition= {(Wc- Wt) × 100 }/Wc (1)
The mice were divided into five groups (n-5). The basal rectal temperature was measured by a digital thermometer (Model MT-101, N & B Medical). The mice were administered 20% yeast suspension with subcutaneous treatment and 4 h later, the rectal temperature of the hyperpyrexic mice was measured. Saline, paracetamol (1.3mg/20g BW), and EESL (4.2, 6.3, and 8.4mg/20g BW), were orally administered to the animals. The rectal temperature was measured for 4 h with a range every 1 h. The last temperature was compared with pre-treatment temperature (temperature taken at 4 h post yeast suspension injection)7.
Results were presented as the mean ± standard deviation (SD). One-way ANOVA was conducted for statistical analysis, followed by Tukey-Kramer post hoc test.
liberation of endogenous substances such as serotonin histamine, prostaglandins, and bradykinins, which stimulate sensory nerve ending20. The abdominal constrictions response is hypothesized cause by local peritoneal receptors21. This acid will increase the levels of PGE2, PGF2α, and lipoxygenase products in peritoneal fluids22.
Figure 1. Analgesic activity results (n = 5)
Mefenamic acid is NSAID, which inhibit prostaglandin synthesis, by binding to and blocking prostaglandin receptors on cells23. Pain response is characterized by the number of writhing every 5 min for 60 min (Figure 1). More writhing showed a small analgesic activity. The analgesic activity by EESL may be via central or peripheral mechanisms. The analgesic activity of EESL was dose-dependent, higher dose will produce higher activity. EESL possessed analgesic activity to acetic acid-induced pain in mice, their activities were similar to mefenamic acid. EESL 8.4 mg/20 g BW showed the same analgesic activity as mefenamic acid (Figure 1). There was significantly different analgesic activity between groups (p = 5.49 x 10-8).
Table 1. The protection and effectiveness of the analgesic activity test
|
Group |
% protection |
% effectivity |
|
Mefenamic acid |
59.68 ± 0.19 |
0 |
|
EESL 4.2 mg/20 g BW |
6.66 ± 0.16 |
11.36 ± 0.14 |
|
EESL 6.3 mg/20 g BW |
23.50 ± 0.13 |
39.75 ± 0.16 |
|
EESL 8.4 mg/20 g BW |
39.21 ± 0.18 |
65.68 ±0.15 |
The % protection was calculated from the ratio of the number of writhing between sample to negative control for 60 min and the % effectivity was calculated from the ratio of the number of writhes between sample to positive control for 60 min (Table 1). There was significantly different from % protection between groups (p = 8.21 x 10-8) and % effectivity (p = 5.32 x 10-9).
The analgesic activity in plants with different mechanisms is contributed by alkaloids24-28, flavonoids28- 32, saponins6,28,33, and tannins28,33. We suggested that alkaloids, flavonoids, and saponins, were contributed in the analgesic activity of EESL. This result was consistent with other studies. Alkaloids produce visceral antinociception34. Flavonoids and related compounds (polyphenols) have analgesic effects with a different mechanisms, such as inhibit inflammatory and neuropathic pain, inducing the nitrogen oxide production, and endogenous opioid-dependent mechanisms. Saponins inhibit central and peripheral pain mediators6,35.
The range of mice’s normal temperature was 36.10- 36.50°C (n = 25), which met the standard mice body temperature, i.e. 35.80-37.00°C10. The pyrexia was initiated by yeast, due to inducing the hypothalamus PGE2 production, which set the thermoregulatory center at a higher temperature36,37. Yeast increases the mice body temperature from normal (36.32±0.10°C) to 37.87
± 0.24°C after 4 h of yeast injection.
Figure 2. Antipyretic activity results (n = 5)
The antipyretic activity of paracetamol cause by inhibiting PGE2 synthesis, which inhibits specific COX isoform selectively in the CNS23. The inhibition of PGE2 synthesis was postulated as a mechanism for the antipyretic activity of ethanolic extract38. Paracetamol has reduced the body temperature, from 38.06±0.23°C to 36.20±0.35°C after 4 h of drug treatment. EESL possessed antipyretic activity in yeast-induced pyrexia in mice, their activities were similar to paracetamol. EESL 6.3mg/20g BW showed the same antipyretic activity as paracetamol (Figure 2).
The antipyretic activity of EESL was slightly dose- dependent, a higher dose will produce a slightly higher activity (Figure 2). There was significantly different antipyretic activity between groups (p = 4.59 x 10-9). The antipyretic activity in plants is contributed by alkaloids39, steroids, flavonoid40-44, tannins, triterpenoids, and coumarin glycosides40. We suggested that all secondary metabolites in EESL, i.e. alkaloids, flavonoids, saponins, monoterpenoids, and sesquiterpenoids were contributed in the antipyretic activity of EESL. This result was consistent with other studies. The antipyretic activity of alkaloids was through inhibition of prostaglandin formation39. Saponins and flavonoids are suggested to have pharmacological activity due to work synergistically45, which is involved in inhibition of PGE2 synthesis. Flavonoids suppress TNF-α due to expanding the antipyretic activity46, while its related compounds (polyphenolics) inhibit the arachidonic acid peroxidation, which reduces the prostaglandin levels due to expand fever reduction47. Further investigation is required to isolate the active compound, which is responsible for analgesic and antipyretic activities, and to elucidate the mechanisms of action.
Ethanolic extract of sappan wood leaves has analgesic and antipyretic activity.
The authors declare no conflict of interest.
The authors thank to Yenny Setiayati and Fany Komala Dewi for technical assistance.
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Received on 22.07.2020 Modified on 18.11.2020
Accepted on 05.01.2021 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(10):5213-5216.
DOI: 10.52711/0974-360X.2021.00907