Luvunga sarmentosa, commonly known as saluang belum, is widely used in Kalimantan to relieve pains, rheumatism, boost the immune system, and fever. The research on the free radical scavenging and analgesic effect of the L. sarmentosa stem extract has not been reported. This study aimed to evaluate the free radical scavenging and analgesic activity of the ethanol extract of L. sarmentosa. The L. sarmentosa stem was extracted using 70 ethanol and tested for free radical scavenging using the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and analgesic activity, acetic acid-induced writhing test, and hot plate test in an animal model. The results showed that the 70 ethanol extract of the L. sarmentosa had an anti-free radical scavenging and analgesic activity. The extract has weak free radical scavenging with an IC50 value of 293.45 ug/mL. Analgesic activity using the writhing test indicated that the extract significantly reduced the writhes count after oral administration in a dose-dependent manner compared to the negative control. Extract at a dose of 550 mg/kg BW can reduce the writhing test by 67.60 compared to others. In contrast, the diclofenac sodium reduced the number of writhes by 74.74. While in a hot plate, the extract at a dose of 550 mg/kg BW produced a maximum possible analgesia (MPA) of 17.64, lower than the MPA of diclofenac sodium (51.01). Analgesic activity of the extract has higher inhibition on the writhing test than on the hot plate. The extract could be responsible for the peripheral mechanism by inhibiting the prostaglandin biosynthesis.

Pain is abody's defense mechanism that reacts to stimuli to avoid further tissue damage1. Pain can also be defined as pathological conditions that arise due tofree radicals and oxidative stress in body cells2. The body produces free radicals such as reactive oxygen species (ROS) andreactive nitrogen species (RNS) in endogenous systems when exposed to variousphysiochemical or pathological conditions. Excessive ROS production causesoxidative stress, a process that can damage cell structures, including lipids,proteins, and DNA3. Pain isan unpleasant sensory and emotional experience associated with actual orpotential tissue damage4. However, pain isbeneficial to the immune system. Nevertheless, it causes much suffering anddiscomfort to the victims, lowering the quality of life, disability, ormortality in several cases. Therefore, pain needs to be managed5.

The onset ofpain makes a person look for treatment to reduce pain. Attempts to reduce thepain are with analgesics drug6. Analgesics are substances that can reduce or dispel pain without losingconsciousness. Evaluation of analgesic drugs in public hospitals in China from2013 to 2018 showed an increase in analgesic drugs every year. From 2013 to2018, NSAIDs' annual clinical drug dose increased by about 0.6 times7. However, prolonged use of these NSAIDs produces significant side effectsand are toxic to the liver, kidney, gastrointestinal linings, and reducedauditory ability8. As such, research to discover other alternatives to treat pain iscrucial.

Medicinalherbs have been used for centuries for therapeutic purposes. Many of theseherbs with analgesic activity had been used without any side effects and at alower cost9. World Health Organization (WHO) estimates that more than 80% of the worldpopulation relies on traditional medicines, and the market is rapidly growing.Saluang belum (Luvunga sarmentosa (BI.) Kurz) belongs to the family ofRutaceae, which develops and spreads in the tropical forests of Kalimantan10. Several studies have been carried out to identify the phytochemicals of L.sarmentosa. Its leaves contain apotirucallane triterpenoids named luvunginsA-G, 1α-acetoxyluvungin A, coumarins ostruthin, and8-geranyl-7-hydroxycoumarin, and triterpenes friedelin, flindissone, melianone,niloticin, and limonin have been isolated11.

In CentralKalimantan, this plant was prescribed traditional medication to increasestamina and antioxidants10. The part of the stem, root, and leaves of L. sarmentosa is used intraditional herbal recipes. The stem and root were consumed three times a dayby boiling or brewed with hot water and consumed once a day while warm12,13. The traditional healers prescribed the stem more to treat soreness,fatigue, or pains. Thus far, research on the analgesic activity of L.sarmentosa stem has not been widely reported. Therefore, this study wasconducted to determine the analgesic and free radical scavenging activity of70% ethanol extract of L. sarmentosa stem. The results study willprovide scientific-based evidence on the use of stems in traditional medicine.

The stems of L.sarmentosa was collected in September 2019 from traditional healers inRakumpit District, Palangka Raya, Central Kalimantan, Indonesia (Figure 1). A licensedbotanist at Purwodadi Botanical Garden, East Java, Indonesia, conductedauthentification and identification of the plant with voucher specimen1048/IPH.06/HM/IX/2019. Diclofenac sodium was used as a standard drug foranalgesics and vitamin C for antioxidants. Both of them were obtained from PT.Kimia Farma Tbk, Indonesia. Other materials used were distilled water, aceticacid, carboxymethyl cellulose sodium (CMC-Na 0.5%),1,1-diphenyl-2-picrylhydrazyl radical (DPPH), and 70% ethanol.

Figure1. Luvunga sarmentosa at the forest (a) andthe stem simplicia package from the traditional healer (b)

Plant extraction

The stemof L. sarmentosa was shade dried and powdered mechanically. The powdered(400 g) was macerated in ethanol 70% three times every 24 hours. The extractwas then filtered and concentrated with a vacuum evaporator, then dried with afreeze dryer to obtain a dry extract.

DPPH radicalscavenging assay

The freeradical scavenging activity of the extracts was tested by DPPH radicalscavenging assay. The extract was diluted with methanol at 1000; 800; 600; 400;200; 100; 50; 25; and 12.5 µg/mL. Meanwhile, vitamin C as standard was dilutedat 100; 50; 25; 12.5; 6.25; 3.12; 1.56; 0.78; 0.39; 0.19; 0.095; and 0.0475µg/mL. A solution of 0.25 mM DPPH in methanol was prepared, and 100 µL of thissolution was mixed with 100 µL of extract/standard in methanol at differentconcentrations. The reaction mixture was incubated in the dark at roomtemperature (26°C) for 30 minutes. The experiment was carried out with threereplications, and the absorbance was observed at a wavelength of 517 nm. DPPHradical scavenging activity was calculated using equation [1], in which A0was the absorbance of the control and A1 was the absorbance of theextract/standard.

… [1]

Experimental animal

Malemice (Deutschland, Denken, and Yoken strains) weighing 25-30 g and aged 4-8weeks were obtained from Farma Veterinary Center, Surabaya, Indonesia. Allanimals were maintained on a standard animal pellets diet and water ad libitumat the Animal Laboratory of the Institute of Tropical Disease, UniversitasAirlangga, Surabaya. All the animals were acclimatized for seven days to thelaboratory conditions before the experiment. All animal protocols werecritically reviewed and approved by the Faculty of Veterinary Medicine,Universitas Airlangga, with approval number 2.KE.117.03.202.

Analgesic activityin animal model with acetic acid-induced writhing test

Thirtymale mice were randomly divided into five groups, and each group consisted ofsix mice. Group 1 was treated with carboxymethyl cellulose (CMC-Na 0.5%) as anegative control, group 2 was treated with diclofenac sodium as a positivecontrol at a 40 mg/kg BW, and groups 3, 4, and 5 were treated with ethanolextract of L. sarmentosa at a dose of 50, 300, and 550 mg/kg BW, equalto the dose of 10, 40, and 80 g of simplicia, respectively. All treatments wereadministered orally. The extract and the standard drug were treated 30 minutesbefore 1% acetic acid injection at a dose of 10 mL/kg BW intraperitoneally.After five minutes, each group of mice was observed for the number of writhesfor 45 minutes. The mean value for each group was calculated and compared withthe control. The percentage of analgesic activity was calculated using theequation [2], in which W is thenumber of writhing, c is the negative control, and t is the test14,15.

…[2]

Analgesic activityin animal model with hot plate test

Theanalgesic activity was also evaluated using the hot plate method15-17. Mice were givenoral therapy according to groups. After 30 minutes of treatment, theexperimental animals were placed on a hot plate maintained at 55°C within therestrainer. The reaction time (in seconds) or latency period was determined asthe time for the rats to react to the thermal pain by licking their paws orjumping. The reaction time was recorded before treatment (0 minutes), then 30,60, 90, and 120 minutes after administering the treatments. The maximumreaction time was fixed at 20 seconds to prevent any injury to the tissues ofthe paws. The maximum possible analgesia (MPA) was calculated using theequation [3]18.

… [3]

Data analysis

The results of thestudy were presented in mean±SEM. Statistical analysis was used one-way ANOVAfollowed by post hoc Dunnett’s test for multiple comparisons (GraphPad Prism7.0, Co., Ltd., San Diego, US). The difference between groups was consideredsignificant at a p-value <0.05.

Thisstudy was conducted to determine the free radical scavenging and analgesicactivities of L. sarmentosa stem ethanol extract. The L. sarmentosawas carried out using 70% ethanol as a solvent by the maceration method. Theextraction yielded 3.9% w/w dry matter and was light brown.

DPPH radicalscavenging assay

DPPH radicalscavenging model is the widely used method to evaluate the anti-free radicalactivity of natural compounds and plant extracts. The results showed thatextract had to scavenge the free radical, with an IC50 value of293.45 µg/mL (Figure 2), while vitamin C had an IC50value of 11.39 µg/mL (Figure 3). The extract was weak, andvitamin C was highly active as a free radical, based on Marjoni and Zulfisa19. The scavengingactivity shows that extract and vitamin C could provide a hydrogen atom to theDPPH radical. The DPPH would oxidize and be decolorized. Stable free radicalDPPH could accept an electron or hydrogen radical to become stable. Itssolution appears a deep violet color. As this electron becomes paired off, theabsorption vanishes, resulting in decolorization20.

Figure2. The IC50 value forradical scavenging activity of L. sarmentosa extract

Figure3. The IC50 value forradical scavenging activity of vitamin C

Analgesic activity withacetic acid-induced writhing test

The aceticacid-induced writhing test has widely been used for screening analgesic drugs14. The writhing testis used to assess peripheral acting analgesics. In the writhing test, theacetic acid injection causes pain by releasing serotonin, histamine,prostaglandins, and bradykinin from arachidonic acid through cyclooxygenase(COX) enzymes1,14,16. The synthesis ofendogenous substances induces contraction of the abdominal muscles that touchthe floor, pull the legs back, and stretch the body21-23.

Analgesic activityin this study indicated that the 70% ethanol extract of L. sarmentosastem significantly reduced the writhes count after oral administration in adose-dependent manner compared to the negative control. After forty-fiveminutes of the test period, the extract at 550 mg/kg BW demonstrated thehighest analgesic activity by reducing the number of writhes by 67.60%, while300 mg/kg BW reduced the number of writhes by 49.30%. The 50 mg/kg BW dosereduced the writhes by 33.28%. However, diclofenac sodium reduced the writhesby 74.74%—the analgesic activity of 70% ethanol extract of L. sarmentosastem presented in Table I.

TableI. Analgesicactivity of 70% ethanol extract of L. sarmentosa stem in aceticacid-induced writhing test

Data were reported as mean ± SEM and analyzed by ANOVAfollowed by Dunnett’s multiple comparison test. * indicate astatistically significant value from negative control, ****p<0.0001.

Analgesic activity withhot plate test

The hot plate is astandard method for evaluating central analgesic activity in animal models thatuse thermal stimuli as pain inducers with temperature was maintained at 55°C.The principle of this method is a change in spinal cord level, whicheffectively describes the centrally mediated anti-nociceptive response24. The paw-licking orjumping are defined as pain reflex behavior25-27. The analgesicactivity of the 70% ethanol extract of L. sarmentosa stem using the hotplate was presented in Table II and Figure 4.

The treatment of 70%ethanol extract of L. sarmentosa stem (50–550 mg/kg BW) and diclofenacresulted in a significant dose-dependent increase in the reaction time tothermal stimulation compared with the negative control. The MPA value ofextract did not show any analgesic effect 60 minutes after treatment butincreased at 90 minutes and declined after that. The highest increase inreaction time was observed with a 550 mg/kg BW dose at 90-minutespost-treatment (17.64%). Diclofenac sodium elicited significant analgesicactivity within 30 minutes following administration, as evidenced by thegradual increase throughout the observation period. At the peak of activity (90minutes), diclofenac sodium showed an MPA of 51.01%.

TableII. Analgesicactivity of 70% ethanol extract of L. sarmentosa stem in hot plate test

Data were reported asmean ± SEM; n=6. One-way ANOVAwas carried out using Dunnett’s multiple comparison test. *indicate astatistical significant: *p <0.05; **p <0.01; ***p<0.001; ****p <0.0001

Writhing responseinduced by acetic acid and hot-plate latent pain response in mice are twocommon and important models for screening analgesics. These two models couldcause pain by liberating PGs and many others that excite pain nerve endings.Both methods were used to study ethanol extract's peripheral and centralanalgesic activities. The results showed that ethanol extract has higherinhibition on the acetic acid test than the hot plate. This observation pointsout that extract possesses peripherally-mediated antinociceptive propertiesthat may work via reducing the level of prostaglandin synthesis or otherinflammatory mediators, which is much like diclofenac. Diclofenac sodiumperforms its action in peripheral acting by inhibiting the synthesis of prostaglandins(pain mediators) by inhibiting COX-1 and COX-215. The analgesicactivities of 70% ethanol extract of L. sarmentosa stem could beattributed to one or more phytochemical compounds present in the extract.

The phytochemicalscreening in this study showed that 70% ethanol extract of L. sarmentosastem contained terpenoid and flavonoid compounds. Their compounds in theextract may contribute to the anti-free radical and analgesic activity.Flavonoids are polyphenolic compounds that can change or reduce free radicals28. Triterpenoids orsteroids are compounds that have a role as antioxidants. The antioxidantmechanism of triterpenoids is by scavenging reactive species, such assuperoxide and metal chelating29. Besides that, aflavonoid is also known to have analgetic activity30. Flavonoids canalso reduce arachidonic acid production by inhibiting neutrophils'degranulation31.

Figure4. Maximum possible analgesia ofL. sarmentosa stem extract compared to diclofenac sodium evaluated byhot plate test

The70% ethanol extract of L. sarmentosa stems had weak anti-free radicalactivity. However, it exhibitedsignificant analgesic activity, possibly by a peripheral pain mechanisminhibiting the prostaglandin pathway.

The authors are gratefulto Universitas Airlangga for the funding through the Faculty of PharmacyExcellent Research (Penelitian Unggulan Fakultas Farmasi), contract no. 989/UN3.1.5/PT/2021.

All authors have anequal contribution in carrying out this study.

None.

Theauthors declare no conflict of interest.

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