SECONDARY METABOLITES, IN-VITRO ANTIOXIDANT AND ANTIULCER POTENTIAL OF THE PHYLLOCLADE EXTRACT OF MUEHLENBECKIA PLATYCLADA (F. MUELL) MEISN

SECONDARY METABOLITES, IN-VITRO ANTIOXIDANT AND ANTIULCER POTENTIAL OF THE PHYLLOCLADE EXTRACT OF MUEHLENBECKIA PLATYCLADA (F. MUELL) MEISN

Byju Anju and N. Geetha *

Department of Botany, Bharathiar University, Coimbatore, Tamil Nadu, India.

ABSTRACT: The present study aimed to determine secondary metabolites, in-vitro antioxidant, and antiulcer activities using various solvent-based Muehlenbeckia platyclada extracts. Solvents based phylloclade extraction was used to determine various secondary metabolites such as phenols, flavonoids, terpenoids and tannins and to assess in-vitro antioxidant activities such as DPPH, ABTS^.+, superoxide, hydrogen peroxide, nitric oxide, FRAP, Phosphomolybdenum, metal chelating and reducing power activities. Further, in-vitro antiulcer activities such as α-chymotrypsin enzyme, urease enzyme inhibitory activity, H⁺ K⁺ ATPase activity and acid neutralization capacity (ANC) were performed, and the resultant was analyzed using response surface methodology (RSM) to develop an adequate functional relationship between a response of interest (ME) with independent variables (Time, HCL consumed and NaOH consumed). A correlation coefficient was made between secondary metabolites and in-vitro antiulcer activities. Methanolic extract (ME) showed higher levels of secondary metabolites, in-vitro antioxidant activities and was subsequently selected to evaluate its antiulcer potential. A significant inhibition for alpha chymotrypsin (88.84%), urease (68.52%), and H⁺K⁺ ATPase (91.75%) was compared with respective standards. RSM results of ME for ANC indicated that 10 min, 22.66 mL HCl and 30.7 mL NaOH are adequate to get maximum ANC i.e. 46 mEq/ml. A significant correlation (P<0.05) was found between phenol and ACE (R²=0.948), between phenol and HKA (R²=0.941) and phenol and UE (R²=0.952). These results suggest that ME of M. platyclada possesses potential antiulcer activities due to higher levels of secondary metabolites and in-vitro antioxidant activities.

Keywords: Muehlenbeckia platyclada, cladode, In-vitro antiulcer potential, In-vitro antioxidant activity, Secondary metabolites, Methanolic extract

INTRODUCTION: Phytocompounds from plants have a variety of therapeutic benefits, and they have been employed in managing several human illnesses. Due to numerous side effects and the high cost of chemical pharmaceuticals, there is a renewed interest in natural medicines in the form of standardized plant extracts ¹.

M. platyclada (F. Muel.) Meisn, a member of the Polygonaceae family that is used as a common natural remedy by many ethnic groups ², is endemic to the Southern Hemisphere.

The plant is commonly known as centipede, tapeworm, or ribbon bush with several medicinal properties like anti-inflammatory, analgesic, anti-tumor, anti-HIV, anti-diarrheal, anti-fungal, anti-hepatic, anti-lipolytic, antioxidant, immuno-stimulant and antiulcer ^{2, 3}. According to several studies, phylloclade is a good source of flavonoids ⁴. Secondary metabolites in plants combine with nutrients and fibres to form an integrated defense mechanism that protects against illness and stress ⁵. Under any type of stress, living system produce more reactive oxygen species (ROS) than enzyme and non-enzyme-based antioxidants, such as superoxide anion radicals, hydroxyl radicals and hydrogen peroxide. Cell damage is brought by an imbalance between free radicals, antioxidants ^6-9 and various diseases ^{10, 11}. Supplementing the diet with antioxidants from natural plant sources is the way to address this issue ¹². Peptic ulcers and ulcerative colitis are the most common gastrointestinal tract lesions. Peptic ulcer disease, which affects many individuals worldwide, occurs when some aggressive and gastroprotective factors are in an unbalanced state. Antacids, weak bases, work to lessen gastric acidity by interacting with it to produce salt and water. They have been shown to have good antiulcer properties; extended use can cause several adverse effects, including diarrhea, renal failure, and constipation ¹³.

The H. pylori bacteria release the urease enzyme to defend themselves from the stomach's acidic environment. This enzyme converts urea into ammonia, which protects bacteria from the stomach's acidic environment and facilitates their survival ¹⁴. One effective strategy for eliminating stomach bacteria is to inhibit urease activity ¹⁵. Due to their toxicity and less stable chemical and physical features in the natural environment, synthetic medicines cannot be used to modulate urease function ¹⁶. Plant-derived urease inhibitors have great efficiency against microorganisms while less harming human cells ¹⁴.

Therefore, the finest antiulcer medications could be derived from plants with urease inhibitory capability ^17-23. One of the proteolytic enzymes chymotrypsin, is released into the duodenum by the pancreas. The most potent inducer for the secretion of chymotrypsin and the release of gastrointestinal hormones occurs when food enters the duodenum in the form of partially digested chyme ²⁴. When compared to the healthy control groups, illness groups like those with duodenal and stomach ulcers showed a considerable increase in chymotrypsin activity ²⁵.

Hyperchlorhydria is a stomach situation characterized by uncontrolled hypersecretion of hydrochloric acid from the parietal cells of gastric mucosa through the proton pump ²⁶.

It is known that the proton pump enzyme H⁺ K⁺- ATPase transports H⁺ against a concentration gradient in stomach parietal cells, where it is in charge of producing acid. Various natural products of plant origin i.e. Garcinia kola ²⁷, Cissus quadrangularis ²⁸, Acalypha wilkesiana ²⁹, Azadirachta indica ³⁰ and Hypericum perforatum ³¹ were found to have the inhibitory potential for H⁺/K⁺ ATPase. Acidity is a common gastrointestinal problem due to extreme secretion of gastric acid or stomach acid which worsens the stomach lining mucosa and produces ulceration ³². Antacids are general drugs that reduce or neutralize gastric acid, thereby decreasing gastric pH ³³. Acid neutralization capacity (ANC) is one of the mechanisms for controlling acidity of the stomach ³⁴ and it is defined as the number of moles of acid neutralized by given antacid ³⁵. ANC screening using various plant extracts has been reported earlier ^36-40.

This assay comprises three independent variables, such as consumed volume of HCl, NaOH and Time which are used to determine the end points of the titration during an experiment concerning ANC of a given antacid. Response surface methodology (RSM) is a valid statistical tool to standardize the experimental procedure by applying and studying the effect of different experimental variables and their interactions on the outcome of the result by decreasing the number of experiments ⁴¹.

Many studies have proved that RSM is an efficient technique for the optimization of various experimental protocols including standardization of plant extraction process for various in-vitro antioxidants (Vitis vinifera ⁴² and Origanum vulgare ⁴³ optimized condition for enhanced production of alpha galactosidase enzyme (Fusarium moniliforme) ⁴⁴, normalization of ultrasonic assisted extraction of flavonoids (Crinum asiaticum) ⁴⁵ and selection of specific plant tissue culture media (Berberis vulgaris) ⁴⁶. The present study was designed to investigate secondary metabolites, in-vitro antioxidants and anti–ulcer potential of different solvent-based phylloclade extracts of M. platyclada.

MATERIALS AND METHODS:

Plant Material Collection, Authentication and Preparation of Extracts: M. platyclada was collected from Kerala and authenticated by the Botanical Survey of India, Coimbatore (BSI/SRC/5/23/2019/Tech. 3431). Phylloclade was cleaned, dried under shade, and ground into fine powder. For the cold plant extraction process, the powder was treated with various solvents (petroleum ether, benzene, chloroform, acetone, methanol, and water) successively based on their polarity in 1:10 ratio for 24 h on an orbital shaker (180 rpm) at room temperature.

The filtrate was concentrated through evaporation at room temperature and dissolved in their respective solvents to get the working solution of 10 mg/ 10 mL (w/v).

Quantitative Determination of Secondary Metabolites: All the various solvent-based phylloclade extracts of different concentrations (0.1-0.5 mg/mL) were subjected to quantitative analyses of different secondary metabolites such as total phenols, flavonoids, tannins and terpenoids.

Determination of Total Phenols: For the determination of total phenolic content, different concentration of extracts were placed in test tubes and made up to a volume of 1 mL with distilled water. Then 0.5 mL of Folin–Ciocalteu reagent (1:1 with water) was added.

After five minutes of incubation, 2.5 mL of sodium carbonate solution (20%) was added, the reaction mixture was vortexed and incubated in the dark for 40 min, and the absorbance was recorded at 760 nm. The amount of total phenols was calculated as Gallic acid equivalents from the calibration curve ⁴⁷.

 Determination of Total Flavonoids: Different concentrations of all the solvent extracts were taken in test tubes and made up to a volume of 1 mL with distilled water. The sample solution was mixed with 0.15 mL of 5% NaNO₂ solution. After 6 min, 0.15 mL of 10% AlCl₃ solution was added and allowed to stand for 6 min and then 2 mL of 4% NaOH solution was added to the mixture. Immediately, water was added to bring the final volume up to 5 mL and then the mixture was thoroughly mixed and kept for 15 min. The absorbance of the mixture was determined at 510 nm using water as a blank. The analysis was performed in triplicate, and the results were expressed in rutin-equivalent flavonoids per gram extract ⁴⁸.

Determination of Total Tannins: Tannin content of the samples were determined using insoluble polyvinyl-polypirrolidone (PVP), which binds tannins ⁴⁹. Different extracts were placed in test tubes, made up to a volume of 1mL with distilled water and mixed with 100 mg PVP, and incubated at 4ºC for 15 min. Then the mixture was centrifuged for 5 min at 8000 rpm. The non-tannin phenols were determined as mentioned for total phenol content using the clear supernatant. Tannin content was calculated by taking the difference between total and non-tannin phenolic contents.

Determination of Total Terpenoids: Solvent extracts with different concentrations were taken and made into 1mL using distilled water. Then, it was mixed with 3 mL of chloroform and allowed to stand for 3 min after adding 0.2 mL of conc. H₂SO₄, it was incubated for 2 h under dark conditions. Then, it was centrifuged and the supernatant was collected. To this, 3 mL of 95% methanol was added and vortexed. The absorbance of the mixture was determined at 538 nm against water as blank. The total terpenoid amount was calculated as linalool equivalents from the calibration curve ⁵⁰.

In-vitro Antioxidant Assays: During secondary metabolites analysis, 100µg/ml concentration showed high secondary metabolites content among various phylloclade extracts. Therefore, all the extracts in this concentration were subjected to different in vitro antioxidant assays such as DPPH, ABTS•+, Superoxide (O2^-), Hydrogen Peroxide (H₂O₂), Nitric Oxide (NO^.), FRAP, Phosphomolybdenum, Metal chelating (MC) and Reducing power (RP) activities.

DPPH Radical Scavenging Activity: A solution of 0.135 mM DPPH in methanol was prepared, and 1.0 mL of this was mixed with different concentrations of phylloclade extracts. The reaction mixture was vortexed thoroughly and left in the dark at room temperature 27°C for 30 min and the absorbance of the mixture was measured spectrophotometrically at 517 nm. Ascorbic acid was used as a standard for reference. The inhibitory concentration (IC₅₀) of several extracts required to decrease the initial concentration of DPPH by 50% was calculated ⁵¹.

ABTS•+ Radical Scavenging Assay: To phylloclade extracts and trolox standards, 1 mL of 7mM ABTS solution was added and allowed to react for 30 min, and the absorbance was taken at 734 nm. The inhibitory concentration (IC₅₀) of several extracts required to decrease the initial concentration of ABTS by 50% was calculated ⁵².

Ferric Reducing Antioxidant Potential (FRAP): The freshly prepared FRAP reagent (900 µL) was mixed with water (90 µL) and phylloclade extracts. The reaction mixture was incubated for 30 min under 37°C, and the optical density was read at 593nm. The antioxidant power was expressed in ferric – TPTZ reducing power corresponding to that of 1mM ferrous sulfate heptahydrate ⁵³.

Determination of Total Antioxidant Capacity by Phosphomolybdenum Assay: The assay is based on the reduction of Mo (VI) – Mo (V) by the extract and subsequent formation of a green phosphate/Mo (V) complex at acid pH. 100 µg/mL phylloclade extracts were combined with 1mL of reagent solution (0.6M sulfuric acid, 2.28mM sodium phosphate, and 4mM ammonium molybdate) and incubated at 95^oC for 90 min. Then the absorbance of the solution was measured at 695nm, and the antioxidant activity was expressed as the number of ascorbic acid equivalents ⁵⁴.

Assay of Superoxide Radical (O2^-) Scavenging Activity: The assay was based on the capacity of the solvent extracts to inhibit formazan formation by scavenging the superoxide radicals generated in riboflavin light–NBT system ⁵⁵. Briefly, phylloclade extracts containing 50mM sodium phosphate buffer (pH 7.6), 20 mg riboflavin, 12 mM EDTA and 0.1 mg NBT were allowed for 90 sec illumination and then absorbance was measured at 590 nm. The percentage of inhibition and IC₅₀ values were determined.

Scavenging of Hydrogen Peroxide (H₂O₂): A solution of hydrogen peroxide (2mM) was prepared in phosphate buffer (pH 7.4) and 100µg/mL solvent-based phylloclade extracts were added to 0.6 mL of H₂O₂. Absorbance was determined at 230 nm after 10 min against a blank solution containing phosphate buffer without H₂O₂ ⁵⁶.

Assay of Nitric Oxide Scavenging Activity (NO): Sodium nitroprusside in an aqueous solution at physiological pH spontaneously generates nitric oxide, and it interacts with oxygen to produce nitrite ions that can be estimated using Greiss reagent. Nitric oxide scavengers compete with oxygen leading to reduced production of nitrite ions. For the experiment, sodium nitroprusside (10 mM) in phosphate-buffered saline was mixed with different concentrations of solvent extracts and incubated at room temperature for 150 min. After the incubation, 0.5 mL of Griess reagent (1% sulfanilamide, 2%H₃PO₄ and 0.1% N-(1-naphthyl) ethylene diamine dihydrochloride was added. The absorbance of the chromophore formed was read at 546 nm. The inhibitory concentration (IC₅₀) of several extracts required to decrease the initial concentration of NO by 50% was calculated ⁵⁷.

Metal Chelating Activity: Initially, 0.1 mM FeSO₄ and 0.25 mM of ferrozine combined and produce a Fe²⁺-ferrozine complex. To this 0.2 mL of the phylloclade extract was added and incubated for 10 min at room temperature. The absorbance of the mixture was recorded at 562 nm and EDTA was used as a positive control ⁵⁸.

Fe ³⁺ Reducing Power Activity: 100 µg/mL plant extracts in 1mL of methanol were mixed with phosphate buffer (2.5 mL,0.2 mol/L, pH 6.6) and potassium ferricyanide [K₃Fe(CN)₆] (2.5 mL, 1%). The mixture was incubated at 50°C for 20 min. A portion (2.5 mL) of trichloroacetic acid (10%) was added to the mixture and then centrifuged for 10 min. The upper layer of solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl₃ (0.5 mL, 0.1%), and the absorbance was measured at 700nm. Increased absorbance of the reaction mixture indicates increased reducing power⁵⁹.

In-vitro Antiulcer Assays: Based on higher levels of secondary metabolites and potential in-vitro antioxidant activities, plant methanolic extract was selected for various in-vitro antiulcer assays.

α – Chymotrypsin Assay: A total volume of 100 μL assay mixture contained 60 μL Tris-HCl buffer (50 mM pH 7.6), 10 μL methanolic extract, and 15 μL (0.9 units) purified chymotrypsin enzyme. The contents were mixed and incubated for 20 min at 37^oC and pre-read at 410 nm. The reaction was initiated by the addition of 15 μL (1.3 mM) substrate (N-succinyl phenyl-alanine-P nitroanilide). The change in absorbance was observed after 30 min at 410 nm. All reactions were performed in triplicates. Chymostatin (0.5 mM) was used as a ‘positive control ⁶⁰.

Percentage of inhibition = [𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑐𝑜𝑛𝑡𝑟𝑜𝑙) − 𝐴𝑐𝑡𝑖𝑣𝑖𝑡𝑦 (𝑠𝑎𝑚𝑝𝑙𝑒)] × 100

Where activity (control) is the control absorbance and activity (sample) is the sample absorbance

 Urease Inhibition Assay: Nessler’s reagent 61 determined the urease inhibition assay. A solution consisting of 0.25 mL urease (0.1 mg/mL) from jack bean, 2 mL of Tris – HCl buffer (pH 8.0) and 0.25 mL urea (60 mM) were incubated with 100 µL of the methanolic extract at 30^oC for 20 min. After incubation, 1 mL of 10% trichloroacetic acid and 0.5 mL Nessler’s reagent were added to stop the reaction. The absorbance was measured at 436 nm and thiourea was used as the standard inhibitor.

The percentage inhibition was calculated from the formula:

IC (%) = (1 – 𝑇 / 𝐶) × 100

Where IC (%) is the inhibitory activity of UE. T is the absorbance of the test sample and C is the absorbance of the control sample

H+ K+ ATPase Inhibition Assay:

Preparation of H⁺ K⁺-ATPase Enzyme: To prepare H⁺ K⁺-ATPase enzyme, fresh goat stomach was obtained from a local slaughterhouse at Coimbatore, Tamil Nadu, India (IAEC No: KMCRET/ReRc/Ph. D/19). The stomach was cut open, the gastric fundus mucosa was cut off, and the inner layer was scraped out for parietal cells ⁶¹. Thus, obtained cells were homogenized in 16 mM Tris buffer (pH 7.4) containing 10% Triton X-100 and centrifuged at 6000 rpm for 10 min. The supernatant (enzyme extract) was used to determine the H⁺, K⁺- ATPase inhibition.

Assessment of H⁺, K⁺- ATPase Inhibition Assay: The reaction mixture containing 0.1mL of enzyme extract and methanolic cladode extract (100µL) was pre-incubated for 60 min at 37°C. The reaction was initiated by adding substrate 2 mM ATP (200 μL). 2 mM MgCl₂ (200 μL) and 10 mM KCl (200 μL) were added and incubated for 30 min at 37°C. Then the reaction was stopped by the addition of assay mixture containing 4.5% ammonium molybdate and 60% perchloric acid followed by centrifugation at 2000 rpm for 10 min and inorganic phosphate released was measured spectrophotometrically at 660nm ⁶². Omeprazole (0.5mM) was used as a positive control.

Percentage of enzyme inhibition was calculated by using the formula;

Percentage of inhibition = [Activity (control) − Activity (sample)] × 100

Where activity (control) is the control absorbance and activity (sample) is the sample absorbance.

Application of RSM to Optimize Different Parameters of ANC: Results obtained for ANC was analyzed using RSM to develop an adequate functional relationship between a response of interest (ME) and some independent variables (Time, HCl consumed and NaOH consumed for ANC).

Experimental design and Optimization: The RSM with CCD (Central Composite Design) was selected to optimize the process variables for enhanced ANC. The experimental design comprised 20 experimental runs with 14 factorial and 3 axial points (α), respectively at a distance of ±2cm from the center and 3 replicates of central points. The number of experiments was calculated from the following equation:

N = 2k (factorial points) + 2k (axial points) + n₀ (central points)

N is the total number of experiments, k is the independent variable number, and n0 is the replicate number of the central points, resulting in an experimental design of 20 points. A second order polynomial regression model was used to correlate the relationship between independent variables and dependent variable responses i.e., Time, HCL consumed and NaOH consumed for ANC.

The second order polynomial regression model was as follows:

𝑌 = 𝛽0 + ∑𝛽𝑖𝑋𝑖3𝑖 = 1 + ∑𝛽𝑖𝑖𝑋𝑖2 + ∑∑𝛽𝑖𝑗𝑋𝑖𝑋𝑗 + 𝜀3𝑗=𝑖 + 12𝑖 = 13𝑖 = 1

Where Y is the dependent variable responses for ANC (Y₁), β₀ is the model constant, β_i, β_ii, and β_ij are model coefficients, X_i and X_j are the coded value of independent variables, and ε is an error. Subsequently, additional experiments were carried out to verify the error in the process variables statistically.

Acid Neutralizing Capacity: The acid neutralizing capacity (ANC) of M. platyclada methanolic extract (100µl) was compared with the standard antacid Gelucil (10 mg/mL). Water was added and mixed to the 5 mL plant extract to make the total volume of 70 mL and then 30 mL of 1N HCl was added. It was stirred for 15 minutes and added 2-3 drops of phenolphthalein solution. The excess HCl was immediately titrated with 0.5 N sodium hydroxide solution drop wise until a pink color is appeared ⁶³.

The moles of acid neutralized is calculated by,

Moles of acid neutralized = (𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝐻𝐶𝑙 × 𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 𝑜𝑓 𝐻𝐶𝑙) − (𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑁𝑎𝑂𝐻 × 𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 𝑜𝑓 𝑁𝑎𝑂𝐻)

Acid neutralizing capacity (ANC) per gram of antacid = 𝑀𝑜𝑙𝑒𝑠 𝑜𝑓 𝐻𝐶𝑙 𝑛𝑒𝑢𝑡𝑟𝑎𝑙𝑖𝑧𝑒𝑑 / 𝐺𝑟𝑎𝑚𝑠 𝑜𝑓 𝑎𝑛𝑡𝑎𝑐𝑖𝑑/𝑒𝑥𝑡𝑟𝑎𝑐

Statistical Data Analysis: Statistical analysis was carried out by one-way analysis of variance (ANOVA) test and Pearson’s Correlation Coefficient (R²) using a statistical package program (SPSS 10.0). The mean comparisons were considered statistically significant when p ≤ 0.05. The data obtained from ANC was evaluated through the Minitab software (version 21.1.1, LLC, Inc., 2021 State College, Pennesylvania, USA), and the regression model fit values were assessed based on the R² (coefficients of determination).

RESULTS:

Quantitative Determination of the Secondary Metabolites: The results of total phenols, flavonoids, tannins, and terpenoids are given in Fig. 1. For quantification of these secondary metabolites, different concentrations of various solvent based phylloclade extracts of M. platyclada i.e. 100, 200, 300, 400 and 500 µg/mL were taken. Of these concentrations, 100µg/mL showed high content of all the secondary metabolites analyzed.

Total Phenol Content: Total phenolic content of various solvent-based phylloclade extracts of M. platyclada varied widely and ranged from 65.6 to 610 mg GAE/g extract. Methanolic extract showed higher total phenolic content i.e. 610 ± 4.0 mg GAE/g extract than the other solvent extracts Fig. 1A.

Total Flavonoid Content: Total flavonoid content was found to be high in methanolic extract (615.66 ± 1.52 mg RU/g extract) followed by aqueous and acetone extracts (289.33 ± 2.08 and 260.66 ± 2.51 mg RU/g extract, respectively. Low flavonoid content was observed in petroleum ether extract i.e. 84.0 ± 2.0 mg RU/g extract Fig. 1B.

 Estimation of Tannin Content: Total tannin content of the various extracts was varied, and the range was found from 27.86 to 670.33 mg TAN/g extract. Among various extracts, methanolic cladode extract registered higher tannins, i.e. 670.33±3.05 mg GAE/g extract Fig. 1C.

Estimation of Terpenoid Content: Total terpenoid content of various phylloclade extracts of M. platyclada was observed in the range of 11.0 to 295.66 mg LIN/g extract. Of various extracts used, methanolic extract showed maximum terpenoid content i.e. 295.66±4.16 mg LIN/g extract Fig 1D.

FIG. 1: QUANTITATIVE ANALYSIS OF SECONDARY METABOLITES OF M. PLATYCLADA

In-vitro Antioxidant Activities:

DPPH Radical Scavenging Activity: The data on DPPH^• radical scavenging activity of the various solvent-based phylloclade extracts of M. platyclada along with the best-known natural antioxidant standard, ascorbic acid is presented in Table 1. Low mean IC₅₀ value was found for methanolic extract (5.22 µg/ mL), indicating its potential to scavenge DPPH free radicals compared to standard ascorbic acid (18.59 µg/ mL). Next to the methanolic extract, aqueous extract (5.70 µg/ mL) showed significant activity. Among all solvent-based extracts, petroleum ether extract had higher IC50 value, i.e. 17.59 µg/ mL, indicating its poor scavenging activity.

Total Antioxidant Activity by ABTS^•+ Radical Cation Decolorization Assay: Table 1. shows the ABTS radical scavenging ability of various phylloclade extracts. It can be ranked as methanolic extract (IC₅₀ 1.34 µg/mL) > aqueous extract (IC₅₀1.38 µg/mL) > acetone extract (IC₅₀ 1.37 µg/mL) > chloroform extract (IC₅₀ 1.47 µg/mL) > petroleum ether extract (IC₅₀ 1.9 µg/mL) > benzene (IC₅₀ 3.9 µg/mL).

Nitric Oxide Radical Scavenging Activity: Among various extracts, the methanolic extract showed the highest nitric oxide scavenging activity 1.25 µg/mL IC₅₀ than that of the standard rutin (7.98 µg/mL IC₅₀).

Petroleum ether extract showed a Very low scavenging effect with an IC₅₀ value 2.94 µg/mL Table 1.

TABLE 1: MINIMAL INHIBITORY ACTIVITY OF DIFFERENT SOLVENT-BASED EXTRACTS OF M. PLATYCLADA

Values are expressed as mean± SD of three replicates. Means followed by the different letters. Within the column are significantly different at P˂0.05 level. Standards: 2, 2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) – ascorbic acid, 2, 2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) – Trolox, Nitric oxide (NO^.) – Rutin, Hydrogen Peroxide (H₂O₂) – Rutin, Superoxide radical scavenging activity (O₂^-) – Rutin.

Hydrogen Peroxide Radical Scavenging Activity: Of various extracts analyzed, methanolic extract was capable of scavenging hydrogen peroxide effectively compared to other extracts.

It showed low mean IC₅₀ value (2.1 µg/mL) than the standard (5.78 µg/mL IC₅₀). Next to the methanolic extract, the aqueous extract had 3.5 µg/ml IC₅₀ value Table 1.

Superoxide Radical Scavenging Activity: Among various extracts, the methanolic extract showed better superoxide anion radical scavenging activity i.e., 25.14µg/mL IC₅₀.

Acetone extract shows less inhibitory effect with IC₅₀ value (56.44 µg/mL) for this activity Table 1.

Ferric Reducing Antioxidant Potential (FRAP): FRAP assay measures the reducing potential of plant antioxidants reacting with a ferric tripyridyltriazine (Fe³⁺-TPTZ) complex and producing a coloured ferrous tripyridyltriazine (Fe²⁺-TPTZ). FRAP values of the different solvent extracts of M. platyclada are shown in Fig 2A. Of various extracts, the methanolic extract had the highest FRAP value i.e. 696.66 µM Fe (II) E*/mg extract, followed by aqueous extract i.e. 541.66 µM Fe (II) E*/mg extract. Petroleum ether extract showed the lowest FRAP value i.e. 192.66 µM Fe (II) E*/mg extract. Results indicate that the methanolic extract has the stronger ferric-reducing power than other solvent extracts.

Determination of Total Antioxidant Capacity by Phosphomolybdenum Assay: The total antioxidant activity by phosphomolybdenum assay was based on the reduction of Mo (VI) to Mo (V) in the presence of the antioxidant compound and subsequent formation of a green phosphate/ Mo (V) complex at acidic pH and at a higher temperature. The antioxidant capacity was reported as ascorbic acid equivalents. The methanolic extract exhibited the best antioxidant activity (760.66 mg AAE/g extract) followed by the aqueous extract (464.66 mg AAE/g extract). The results indicated that the methanolic extract contains powerful antioxidants as compared to other solvent extracts Fig. 2B.

 Metal Chelating Activity: The metal chelating ability of the extracts was measured by the ferrous ion ferrozine complex formation. The chelating activity has been mentioned as EDTA equivalents. Each extract interferes with the formation of Ferrous ion (Fe+2) and ferrozine complex, suggesting its metal chelating activity. Among the solvent extracts, methanol shows the highest chelating activity of 272.66 Mm EDTA/g extract, followed by the aqueous extract (225.66 Mm EDTA/g) Fig. 2C.

Reducing Power Activity: A compound's reducing capacity may be a significant indicator of its potential antioxidant activity. The results of the reducing power assay show the highest activity for methanolic extract (798.33 mg RUT/g extract) and the lowest activity for petroleum ether (355.6 mg RUT/g extract) Fig. 2D.

FIG. 2: IN-VITRO ANTIOXIDANT CAPACITY OF M. PLATYCLADA

Correlation Coefficient between Secondary Metabolites and In-vitro Antiulcer Activites: Correlation coefficient was made between secondary metabolites and in-vitro antiulcer activities. Phenol showed significant correlation (P<0.05*) for alpha chymotrypsin (0.948), H⁺ K⁺ ATPase (0.941) and urease (0.952) while flavonoids and tannin had significant correlation only for urease. Terpenoids exhibited maximum linear correlation for urease and acid neutralization capacity Table 2.

TABLE 2:  PEARSON’S CORRELATION COEFFICIENT ANALYSIS BETWEEN SECONDARY METABOLITES AND IN-VITRO ANTIULCER ACTIVITES

Correlation coefficients (R) with the level of significance **=p˂0.01, *=p˂0.05.

In-vitro Antiulcer Activity: During in-vitro antioxidant analysis, the methanolic extract showed better antioxidant and free radical scavenging activity among various solvent-based phylloclade extracts. Therefore, this extract was selected for the in-vitro antiulcer activity.

α-chymotrypsin Assay: Methanolic extract of M. platyclada was tested against α-chymotrypsin enzyme Fig. 3B. The extract showed maximum inhibitory activity (88.84%) for alpha chymotrypsin enzyme compared to the standard chymostatin (42.51%).

Urease Enzyme Inhibition Assay: The urease enzyme inhibition assay was done by utilizing standard thiourea, which showed minimum inhibitory percent (12.84%), whereas methanolic extract showed the maximum percent of inhibition (68.52%) Fig. 3C.

 H⁺ K⁺ ATPase Activity Assay: During H⁺ K⁺ ATPase activity assay, the methanolic extract showed significant (P<0.05) proton pump inhibitory activity in the goat gastric mucosal homogenate Fig. 3A. The inhibitory activity was comparable to the standard drug omeprazole.

The methanolic extract of M. platyclada potently reduced the hydrolysis of ATP by the goat gastric ATPase (91.75%). Omeprazole (100 µg/ml) was used as a positive control, which reduced the activity (67.68%).

FIG. 3: IN-VITRO ANTIULCER ACTIVITIES OF M. PLATYCLADA

Model Fitting of Response Surface Methodology (RSM): Results obtained for ANC were analyzed using RSM to develop an adequate functional relationship between a response of interest (ME) and some independent variables (Time, HCl consumed, and NaOH consumed for ANC). The model CCD was fixed to investigate the effect of Time, HCl, and NaOH consumed for ANC Table 3. These three factors were assessed using the second-order model. The consumed volume of HCl (X₁) and NaOH (X₂) was increased with increasing Time (X₃). When the titration reached its endpoint, the ANC value was found for both the sample and the standard. Table 3 shows the design matrices of the experiments using CCD.

TABLE 3: DESIGN MATRICES OF TIME (X₁), HCL CONSUMED (X₂) AND NAOH CONSUMED (X₃) FOR THE ACID NEUTRALIZATION CAPACITY OF M. PLATYCLADA METHANOLIC EXTRACT AND STANDARD USING CCD DESIGN

BLK: Block, Time (X₁), HCL consumed (X₂) and NaOH consumed (X₃).

The values of the response variables for Time (4.9- 10 min), HCl consumed (16.9-22.6 mL), and NaOH consumed (22.9-30.7 mL). After experimental runs, 22.66 mL HCl, 30.7 mL NaOH and 10 min were found to be required to get maximum ANC Fig. 4.

FIG. 4: RESPONSE SURFACE PLOTS OF M. PLATYCLADA (METHANOL EXTRACT AND STANDARD)

Acid Neutralization Capacity (ANC): The in-vitro acid neutralization effect of the methanolic extract of M. platyclada was compared with the standard antacid gelucil (10mg/mL). ANC of the standard was found to be 65mEq/extract, whereas it was 46mEq/extract for methanolic extract Table 4.

TABLE 4: IN-VITRO ACID NEUTRALIZATION CAPACITY OF M. PLATYCLADA METHANOLIC EXTRACT AND THE STANDARD (GELUCIL)

MEMP: methanolic extract of M. platyclada, Standard: Gelucil. The acid-neutralizing capacity of Standard antacid and methanolic extract of M. platyclada (100µg/ml) was observed in-vitro. The data represent the mean volume of NaOH consumed (mL) and mEq of acid consumed. The methanolic extract shows a maximum ANC (46 mEq /ml) than the standard (65 mEq /ml).

TABLE 5: REGRESSION ANALYSIS AND ANOVA OF MODEL FITTING DESIGN IN ORDER TO EXAMINE THE   STATISTICAL SIGNIFICANCE OF ANC

^aDegrees of freedom, ^bAdjusted sum of squares, ^cAdjusted mean square measures, ^dTest for comparing model variance with residual (error) variance, ^eProbability of seeing the observed F value if the null hypothesis is true.

Table 5 represents the regression analysis and ANOVA of model fitting design to examine ANC's statistical significance. Large F value i.e. 64.91 of ANC indicating the CCD model selected for this analysis was found to be significant. P value obtained (<0.001) in the model response was relatively very low, indicating this model's significance. A large F value and small p-value shows that the independent variables significantly impact the respective response variables. The predicted R – square (Pre. R²) value indicates how well a regression model predicts response values, while the adjusted R – square (Adj. R²) indicates the descriptive power of the regression model while including a diverse number of variables. In this study, the Adj. R² values were found to be 0.845 and 0.889 for ANC of ME and ANC of standard.

DISCUSSION: Ulcerative colitis is a chronic disease that causes the colon to become inflamed regularly. The condition mostly affects the colon and rectum, only damaging the innermost mucosa. The moderate symptoms of the disease include diarrhoea, stomach pain and progressively loose stools. Additionally, when the illness worsens, the patient may develop anemia, weight loss, exhaustion, and loss of appetite. Antacids (which include bismuth for convenience), anticholinergics, H₂-receptor antagonists, and liquorice derivatives are the four categories of peptic ulcer medications. Most commercially available antacids contain magnesium and aluminium but have undesirable side effects, like diarrhoea and constipation. Due to their widespread availability, patient acceptability, efficacy, safety, and low cost, people have used herbal medications for a long time to prevent these issues. Globally, herbal medicine is a well-liked complementary therapy for ulcerative colitis, and hundreds of clinical trials have supported its efficacy. This study aimed to assess the secondary metabolites quantification, in-vitro antioxidants and antiulcer potentials of different solvent-based phylloclade extracts of M. platyclada. During secondary metabolites analysis, among various solvent-based phylloclade extracts, 100µg/ml extract showed high content of all the secondary metabolites analyzed. Therefore, all the extracts in this concentration were subjected to different free radical scavenging assays such as DPPH, FRAP, ABTS•+, phosphomolybdenum, superoxide radical (O2^-), hydrogen peroxide (H₂O₂), nitric oxide (NO^.), metal chelating activity (MCA) and reducing power. Among various plant extracts, ME showed a higher level of in-vitro antioxidant activity.

Polyphenols are one of the abundant plant secondary metabolites and are ubiquitously present in various parts of the plants. Polyphenols reported in M. platyclada are morin-3-O – α rhamnopyranoside, kaempferol – 3 – O – β - gluco-pyranoside, quercetin 3-0-α-rhamnopyranoside and (+) – catechin. The phytocompound quercetin has an effective antiulcer activity that protects and prevents biochemical and morphological changes caused due to the induction of ulcer with ethanol and indomethacin. Kaempferol is a common flavonoid with antioxidant and anti-inflammatory properties with gastric antiulcer effect. Tannins are polyphenols used in the medicinal industry due to their astringent properties. During gastric ulcers, tannins react with the stomach protein and precipitate to form a protective cover, promoting resistance to proteolytic enzymes responsible for ulcer formation. Monoterpenoids are believed to be capable of changing the harmful effect of stress and serve as non-steroidal anti-inflammatory drugs. Lupeol, a pentacyclic terpenoid, possesses gastroprotective activity against ethanol's ulcerogenic effects in the experimental mice. These studies indicate the potential activity of terpenoids against ulcers. The total antioxidant activity determinations were carried out using all the plant extracts of M. platyclada of various extracts, ME showed much antioxidant properties compared to other extracts.

In general, antioxidants act as reducing agents for completely eliminating various free radical intermediates with further inhibition of oxidation. DPPH^• is a stable free radical and it can accept an electron or hydrogen radical to become a stable diamagnetic molecule. In the present study, ME exhibited the highest DPPH^• radical scavenging activity than the other solvent extracts, which was likely due to their higher flavonoid content, the most important phytocompounds for scavenging activity. This trend of DPPH^• free radical scavenging activity had already been well documented in the literature for various plant species.  

M. platyclada phylloclade extracts were found to have effective antioxidants, which was confirmed through various in vitro assays. In FRAP assays, ferric tripyridyltriazine (Fe (III)-TPTZ) becomes ferrous tripyridyltriazine (Fe (II)-TPTZ) through reaction with a reductant under low pH. (Fe (II)-TPTZ exhibits an intense blue colour and can be measured at 593 nm. An increase in absorbance is directly related to the amount of antioxidant present in the reaction mixture. A FRAP value of ME calculated was similar to the reports compared to the standard. ABTS^{. +} assay is an excellent technique for determining hydrogen-donating and chain-breaking antioxidants. In the present study, the efficacy of ME showed higher ABTS^.+ cation radical scavenging activity compared to other extracts. Since ME contains high molecular weight, it can scavenge ABTS more easily^{. +} radicals. In the present investigation, among various extracts, ME significantly showed higher scavenging activity of O₂^.- which is produced in biological systems during cellular respiration and causes damage to biomolecules. Therefore, the removal or neutralization of O₂^{. -} is important to protect the cells from their deleterious effects. NO^. becomes toxic after reaction with oxygen or O₂^.- radicals. Various phenols and flavonoids have been found to neutralize NO^. radicals in previous studies. Several plant extracts have been reported to manifest antioxidant activity by exhibiting high FRAP values in-vitro (Croton caudatos ⁶⁴, Oroxylum indicum ⁶⁵, Garcinia atrovirdis, and Cynometra cauliflora ⁶⁶.

Based on the data, it was understood that MCA was found higher in ME than other extracts. It was evident from the study that ME of M. platyclada possesses a better MCA, which might play a protective note against metal-catalyzed oxidative damage. The reducing power assay evaluates the ability of an antioxidant to donate an electron. In this assay, the ability of extracts to reduce Fe³⁺ to Fe²⁺ was determined. The reducing activities of various extracts of M. platyclada was observed in comparison with the standard. Among all the extracts, ME showed the highest reducing ability.

Herbal medicines play a significant role in treating gastro-duodenal diseases because they have less side effects and are more affordable than synthetic drugs. α-chymotrypsin enables the cleavage of peptide bonds by hydrolysis reaction. In this study, ME of M. platyclada showed more inhibitory activity against α-chymotrypsin than standard chymostatin. Maximum chymotrypsin inhibitory activity was reported in various plants such as Acacia concinna, Caesalpinia bonducella, Lathyrus sativus, Mucura pruriens, psoralea corylifolia and Sapindus mucoross ⁶⁷. Urease (UE) is responsible for the hydrolysis of urea to ammonia and carbamate, the final step of nitrogen metabolism in living organisms. The present study showed profound inhibition of urease by ME compared to standard. H⁺ K⁺ ATPase is a key enzyme in inducing acidity. This investigation studied the potential of ME to inhibit H+ K+ ATPase in-vitro isolated from goat stomachs. The result showed that ME had more significant proton inhibitory activity than standard. In ANC, ME showed a significant reduction (46mEq/extract) compared to standard (65mEq/extract). RSM is an effective statistical tool for optimizing various complex processes simply by saving experimental periods. It is the first report in which RSM tool was utilized to optimize the parameters for the enforced activity of ANC. This statistical tool was applied for the optimization of plant extraction methods for Cynomonium songaricum for prediction of various secondary metabolites biosynthesis from cell suspension culture using yeast extract as elicitor; for optimization of ultrasonic-assisted extraction of flavonoids from Crinum asiaticum ⁶⁸ and optimization of the best conditions such as pressure, temperature and CO₂ flow rate for extraction of triterpenes and sterols from jackfruit seed using supercritical CO₂ extractor. The correlation coefficient analysis between secondary metabolites and the in-vitro antiulcer activities gives significantly correlated values. Many research reports are available about the gastroprotective effects associated with several plant extracts rich in antioxidants ⁶⁹. Several authors have stated the positive correlation between phenolic content and antioxidant potential of various plant extracts.

CONCLUSION: An examination of the phytochemical composition, antioxidant effectiveness, and antiulcer activity of M. platyclada was conducted in this study. Results showed that secondary metabolites such as phenols, flavonoids, tannins, and terpenoids were present in high ME levels, which were further found to have significant in-vitro antioxidant and antiulcer properties. These results suggest that ME of M. platyclada possesses potential antiulcer activities due to higher levels. These experimental data validate the use of this plant in ethnic groups.

Statements & Declarations:

Funding: This work was supported by the University Research Fellowship (URF), Bharathiar University, Coimbatore, Tamil Nadu, India. Grants number:  BU/REG/URF/2021/197.

ACKNOWLEDGEMENTS: The Authors gratefully acknowledge the financial support by Bharathiar University for providing University Research Fellowship.

CONFLICTS OF INTEREST: The authors have no conflicts of interest in financial or commercial issues of the present research work or its publication

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    How to cite this article:

    Anju B and Geetha N: Secondary metabolites, in-vitro anti-oxidant and anti-ulcer potential of the phylloclade extract of Muehlenbeckia platyclada (F. muell) meisn. Int J Pharm Sci & Res 2023; 14(8): 3868-82. doi: 10.13040/IJPSR.0975-8232.14(8).3868-82.

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