The Anti-inflammatory Activity of Helicteres viscida Blume Ethanol Extract in Carrageenan-induced Paw Edema in Mice

Inflammation is a broad and ancient medical term initially referring to a set of classic signs and symptoms, including edema, erythema (redness), warmness, pain and loss of function (stiffness and immobility) (Coleccion, 2014). Inflammation is a “second-line” defense against infectious agents (Stone et al., 2020). Acute inflammation is typical of short duration, occurring before the immune response becomes established and it is aimed primarily at removing the injurious agent. Clinically, acute inflammation is characterized by 5 cardinal signs: redness, increased heat, swelling, pain and loss of function. Redness and heat are due to increased blood flow to the inflamed area; swelling is due to accumulation of fluid; pain is due to release of chemicals that stimulate nerve endings; and loss of function is due to a combination of factors (Chandrasoma et al., 2020). Although inflammatory response processes depend on the precise nature of the initial stimulus and its location in the body, they all share a common mechanism, which can be summarized as follows: 1) cell surface pattern receptors recognize detrimental stimuli; 2) inflammatory pathways are activated; 3) inflammatory markers are released; and 4) inflammatory cells are recruited (Cheng et al., 2017). It starts as the body’s defense reaction, but may tum potentially harmful (Geetika Khanna Bhattacharya., 2016). Because uncontrolled acute inflammation may become chronic, contributing to a variety of chronic inflammatory diseases (Cheng et al., 2017). There are various medicines for controlling and suppressing inflammatory crisis: steroids, nonsteroid anti-inflammatory drugs and immunosuppressants are the practical examples of these medications which are associated with adverse effects while in practice our goal is to apply minimum effective dose by the highest efficacy with the least adverse effects. Consequently, medicinal plants and their secondary metabolites are progressively used in the treatment of diseases as complementary medicine (Mona et al., 2016). 
       
Based on the review of Diegina et al., (2020), the Helicteres genus, Malvaceae, has a pantropical distribution, encompasses about 60 species, with about 149 compounds were isolated and characterized in the genus, being emphasized terpenoids, flavonoids and lignoids. Species belonging to this genus are used for the treatment of various diseases: kidney tonic, diarrhea, dysentery, itching, hematuria, asthma, anti-inflammatory and aroused scientific interest in the search for bioactive compounds present in these plants. These species have demonstrated various pharmacological properties in vitro and in vivo, including insecticide, antidiabetic, antitumor and hepatoprotective activities. The pharmacological potential of Helicteres species has gained prominence, especially with H. isora and H. angustifolia, which have a long history of use in traditional Chinese medicine (Diegina et al., 2020). H. viscida Blume is found in many provinces of Vietnam. In Dak Nong and Khanh Hoa Provinces, it is reported to be used to treat cancer and anti-inflammatory (Vo, 2000). Thus, the present study was conducted to estimate total phenolic, flavonoid content and evaluate the anti-inflammatory of H.viscida Blume against denaturation of protein and induced paw edema in mice from a natural source.

Preparation of extract
 
H. viscida Blume was collected in Dak Nong Province, Vietnam; then dried and grinded to make them powdered after removed the roots. To extracting the organic compounds from the plant powder the percolation method is used based on the protocol of (Phung, 2007) and (Vietnam Pharmacopoeia, 2015) with some modifieds. Plant powder was pre-moistened with ethanol 70% (EtOH 70) for 2 h, then transferred to percolation system and incubated at room temperature for 48 h. The percolation system was opened to to allow the solvent was passed through the sample at the speed of 1-3 ml/min. The collected extract liquid was filtered using paper 125 mm, No. 3 (Whatman), then put in rotary evaporator (Heidolph) at 50^oC, pressure 110 ppm to remove the solvent. The final extract was kept in sterile sample tubes and stored in a refrigerator at 4^oC.
 
Drugs and chemicals
 
Carrageenan and bovin serum albumin (BSA) was purchased from Sigma - Aldrich (St. Louis, USA); diclofenac sodium is a nonsteroidal anti-inflammatory drugs (NSAIDs) was purchased from Institute for Drug Testing (HCMC, Vietnam). All the chemicals powder were diluted with distilled water to experiment concentration and being used immediately after mixing.
 
Determination of the total phenolic contents (TPC)
 
The phenolic content in the extracts was determined based on the standard curve equation of standard phloroglucinol (0 - 100 µg/ml) using the Folin-Ciocalteu assay (Turkmen et al., 2005) with some modifieds. The reaction mixture consists of 50 µl of 100 µg/ml plant extract (diluted with EtOH 70), 250 µl 10% Folin-Ciocalteu solution, 200 µl of 7.5M Na₂CO₃ solution. EtOH 70 was used as a control. The reaction mixture was gently shaken and incubated for 2h at room temperature. The absorbance was measured at 740 nm on ELISA spectrophotometer (Brand….).  Results were expressed as phloroglucinol equivalent, i.e., mg phloro-glucinol /g the extract.
 
Determination of the total flavonoid contents (TFC)
 
The flavonoid content in the extract was determined based on the standard curve equation of standard catechin (0-500 µg/ml) using the aluminum chloride colorimetric assay (Long et al., 2020). The reaction mixture consists of 300 µl of 100 µg/ml plant extract (diluted with EtOH 70), 15 µl 5% NaNO₂ solution, 30 µl of 10% AlCl₃ solution, 100 µl of 1M NaOH solution. Ethanol 70% was used as a control. The reaction mixture was gently shaken and incubated for 5 mins at room temperature. The absorbance was measured at 510 nm on ELISA spectrophotometer. Results were expressed as catechin equivalent, i.e., mg catechin /g the extract.
 
Evaluation of in vitro anti-inflammatory activity by anti-denaturation of protein
 
Anti-protein denaturation of the plant extract was performed on BSA based on the research of Do et al., (2019) and Belkhodja et al. (2024) with some modifieds. The reaction mixture (4 ml) consists of 2 ml of acetate buffer (pH = 5.5), 1 ml of 0.16% BSA solution and 1 ml of plant extract (50-500 µg/ml). NSAIDs was used as a positive control (50-200 µg/ml) while EtOH 70 was used as control for the test. The tubes containing the reaction mixture were incubated at 37^oC for 20 minutes in a water bath (Ellman), then heated up to 70^oC for 5 minutes. After 5 minutes, the test tube containing the reaction mixture was cooled under running water until it cooled and the absorbance was measured at 660nm by spectrophotometer. The percentage inhibition of protein denaturation was calculated by using the following formula:
 

  

       
IC₅₀ values were estimated from the percentage inhibition versus concentration plot, using a linear regression algorithm. The data were presented as mean values ± standard deviation (n = 3).
 
Evaluation of in vivo anti-inflammatory activity by edema induced by carrageenan in mice model
 
To evaluating the anti-inflammatory activity of the H. viscida Blume ethanol extract, paw edema induced by carrageenan is used according to the research of (Sharma et al., 2022) with some modifieds. Healthy Swiss Albino mice aged 7- 8 weeks with an average weight of 20 - 22 gr were supplied from Pasteur Institute (HCMC, Vietnam). They were kept stable for 7 days before doing experiements in laboratory conditions with the temperature around 30±2^oC, humidity 45% - 65%, 12 hours dark-light cycle and the noise was limited. Mice were divided randomly into 6 groups (6 mice/group). Group I is a control group which was oral adminis-tration of 0.9% saline. Group II is a model group which was oral administration of 0.9% saline. Group III is a drug control group which was oral administration NSAIDs 10 mg/kg bw p.o. Group IV to VI is an experimental group which is oral administration plant extract at a dose of 250, 500, 1000 mg/kg bw p.o, respectively. 1h after the oral administration of saline, diclofenac sodium, plant extracts, 25 ml 1% carrageenan was injected to the left hind paw of each animal in group II to VI. The paw thickness was measured at 1, 2, 3, 4, 6 hours after injection to compared with paw thicken at before experiment. The data were presented as mean values ± standard deviation (n = 6).
 
Statistical analysis
 
The data is expressed as the mean ±SD was analyzed by one-way analysis of variance (ANOVA) and Dunnett’s t-test was used as the test of significance. P value <0.05 was considered as the minimum level of significance. Statistical significance is expressed as *P<0.05 or **P<0.01, ***P<0.001, ****P<0.0001 and ns: no statistical significance. All statistical tests were carried out using Grapad Prism version 8.4.3 statistical software.

Total phenolic contents and total flavonoid contents
 
Flavonoids and phenolic compounds are commonly known as plant secondary metabolites that hold an aromatic ring bearing at least one hydroxyl group (Rosa et al., 2020). Flavonoids and phenolic components have been reported on their effective antioxidants, anticancer, antibacterial, cardioprotective agents, anti-inflammation, immune system promoting, skin protection from UV radiation and interesting candidates for pharmaceutical and medical application (Tungmunnithum, 2018). Consequently, the content of total phenolic and flavonoid was played an important role in the initial assessment of the biological activity of medicinal plants. TPC and TFC in H. viscida Blume extract was determined as 44.82±0.29 and 16.95±1.19 mg/g extract respectively. The result was measure base on the standard curve prepared was used for the determination of total phenolic content and flavonoids using different concentrations of phloroglucinol (y = 0.008412x – 0.04257 (R2 = 0,9911)) and catechin (y = 0.001746x – 0.01445 (R2 = 0,990)) respectively. 
 
The anti-inflammatory activity evaluated by anti-denaturation of protein BSA 
 
The in vitro anti-inflammatory activity of plant extract and diclofenac sodium were evaluated upon its ability to protect protein BSA from denaturing by heat. The results are shown in Fig 1 and Fig 2. Based on the 2 figures, the plant extract inhibited protein BSA denaturation increased from 1.98%, 7.29%, 19.88%, 33.12%, 39.44%, 70.58% respec-tively with increasing concentration from 50 to 500 µg/ml, while diclofenac sodium, which is known as a popular antiinflam-matory drug in the market, had shown a higher antiinflam-matory activity - 28.93%, 31.47%, 39.34%, 49.45%, 58.37%, 75.32% respectively to the concentration 50 to 200 µg/ml. Hence, the results suggested that the concen-tration of anti-inflammatory agent correlates with its ability to shield the heat denaturation of BSA protein. The ability to inhibit protein denaturation of the extract and diclofenac sodium with IC₅₀ values was 413.5 μg/ml and 125.1 μg/ml, respectively. The IC₅₀ value was calculated based as the concentration of IC₅₀ as it reached half point of its maximal inhibitory concentration. With a lower the IC₅₀ value, the substance showed a higher protein denaturation inhibition.




       
The anti-inflammatory ability of a biologically active compound is also reflected in its ability to inhibit protein denaturation. Protein denaturation results in inactivation of protein molecules, which is implicated in the formation of inflammatory disorders such as rheumatoid arthritis, diabetes and cancer. Therefore, resistance to protein denaturation may help prevent inflammatory disorders (Rabbani et al., 2018). Thus, when the temperature is high and in a low pH environment, BSA protein are more reactive, easily broken, revealing the hydrophobic groups inherent in the protein molecule, the hydrophobic groups. This does not bind to polar molecules (such as water and buffers) reducing water solubility, increasing viscosity and precipitates clouding the test tube (Varanko et al., 2020). The anti-inflammtory agents can bind to tyrosine, lysine and threonine-rich regions on BSA thereby making the molecule more stable to heat and reducing denaturation and loss of biological activity (Williams et al., 2008). Accordingly from this report, it can be predicted that H. viscida Blume has several components that can bind to the above groups, forming a complex that is stable at high temperature and low pH.
 
Evaluation of in vivo anti-inflammatory activity by edema induced by carrageenan in mice model 
 
The in vivo anti-inflammatory activity of plant extract was evaluated upon its ability to protect paw edema induced by carrageenan. The results are shown in Fig 3 and Fig 4 and there are differences between paw thickness of albino mice through time and between-group experiments. Based on Fig 3, after 6h of experiment, the mice paw in groups II-VI were swollen compared to group I, therefore carrageenan injection to mice left hind foot can induce paw edema. At group II and III, the paw thickness reached its maximum within 1 h (6.05±0.32 and 4.17± 0.27mm, respectively) while at group IV, V and VI paw thickness reached its maximum within 2 h (4.65±0.14, 4.06±0.14, 3.69±0.18 mm, respectively), after reaching its peak, the paw thickness decreased gradually overtime at all the group. At 3h, the paw thickness in group III decreased (3.14±0.45 mm) and there was no statistically significant difference (P = 0.289 > 0.05) compared with the control group (2.70±0.0 5 mm), this result was recorded in group VI at 4h (2.85±0.38 mm) and Group V at 6 h (2.95±0.30 mm) (P = 0.9296 and P= 0.5262, respectively). Meanwhile, in group II and group IV, the paw thickness compared with the control group still had a statistically significant difference (P<0.001). Comparing group II and group IV at 6 h, there is no statistically significant in paw thickness (3.84±0.57 and 3.65±0.29 with P = 0.7576 > 0.05). Thus, the results suggested that mice oral administration of diclofenac sodium (10 mg/kg) can protect paw edema at the early time, while, in mice oral administration of plant extract, doses of 500 and 1000 mg/kg showed protection against paw edema and anti-inflammatory activity, while a low dose of 250 mg/kg showed no protective effect.




       
Inflammation is a complex process involving the transmission and coordination of molecular inflammatory mediators. When there is an inflammatory agent (e.g carrageenan) was injected to the paw, significantly upregulated the levels of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α as well as caused the production and release of NO which is a signaling molecule that plays a key role in the pathogenesis of inflammation (Annamalai et al., 2016) (Necas et al., 2013) (Sharma et al., 2022). The generation of NO is a feature of genuine immune-system cells (dendritic cells, natural killer (NK) cells, mast cells and phagocytic cells including monocytes, macrophages, microglia, Kupffer cells, eosinophils and neutrophils) (Parul et al., 2007). Therefore, large amounts of NO are synthesized, exceeding the physiological NO production by up to 1000-fold (Sharma et al., 2008). The production and release of NO by these NOSs are thought to contribute to tissue injuryand inflammation-induced oedema and hyperalgesi (Necas et al., 2013). The presented of IL-6 induced the production of other inflammatory TNF-á and IL-1â and enhances the formation of LTB4 which, in turn, contributes to the recruitment of PMNs, augments the expression of iNOS and COX-2 protein and activity and ultimately the degree of peroxynitrite formation and tissue injury (Annamalai et al., 2016). Prostaglandins are formed from arachidonic acid by the action of cyclooxygenase (COX) and subsequent downstream synthetases. COX-2, the inducible form, is expressed in response to inflammatory and other physiologic stimuli and growth factors and is involved in the production of those prostaglandins that mediate pain and support the inflammatory process (Simon, 1999). 
       
It is suggestd that the anti-inflammatory agents may have the effect of regulate or inhibited the process of cytokines, acute-phase proteins, prostaglandins, cyclooxygenase (COX)-related metabolites or major immune cell types. previous studies also indicated that NSAIDs Inhibition of cyclooxygenase (COX) enzyme, which takes part in the biosynthesis of prostaglandins (PGs) and thromboxane (TX), therefore coagulation cascade-derived peptides, interleukin (IL)-2, IL-6 and tumor necrosis factor (TNF) (Gunaydin et al., 2018). Plants with contains the small organic organic molecules come from secondary metabolism and have several biological activities and anti-inflammatory actions are highlighted (Nunes et al., 2020).  It is report that Flavonoids are a group of polyphenols with the ability to inhibit the biosynthesis of prostaglandins (Panche et al., 2016), thus it can be suggested that the extract has anti-inflammatory potential through inhibition of cox-2 activity due to the presence of flavonoid groups present in the extract.

Although the mechanisms that H.viscida Blume ethanol extract protect protein BSA from thermal denaturation or its role in pathology that induced paw edema from carrageenan has not been clarified and deeply understood, this study also provides evidence that H. viscida Blume ethanol extract has anti-inflammatory properties through studies in in vitro and in vivo models, thereby narrowing the study field. Thus, further experiments are needed to understand the anti-inflammatory potential of the extract in terms of regulatory and inhibitory pathways. From there, it is possible to conserve, develop and effectively exploit this source of medicinal herbs in the production of quality, safe and reasonably priced drugs to serve people’s health. At the same time, take advantage of the rich source of medicinal herbs in Vietnam.

This work was supported by Dak Nong Department of Science and Technology, Vietnam Academy of Science and Technology.

All authors declared that there is no conflict of interest.