In vivo and in vitro evaluation of pharmacological activities of Adenia trilobata (Roxb.)

Adenia trilobata, locally known as akandaphal in Bangladesh, has some traditional uses. Leaves and stems extracted with pure methanol (MEATL, MEATS) and fractioned by n-hexane (NFATL, NFATS), which was subjected to qualitative phytochemical analysis. The qualitative phytochemical analysis of four extracts showed the presence of secondary metabolites such as alkaloid, carbohydrate, glycosides, flavonoids, phenols, flavonol, and saponins. All four extracts of A. trilobata, exhibited a strong antioxidant activity while a moderately (MEATS = 328 μg/mL) to weakly toxic (NFATL = 616.85 μg/mL) LC50 observed in brine shrimp lethality bioassay. In thrombolytic test, MEATL (18.54 ± 2.18%; P < 0.01) and MEATS (25.58 ± 4.76%; P < 0.0001) showed significant percentage of clot lysis in human blood. The in vivo analgesic activity carried by acetic acid test and formalin test, while the antidiarrheal activity assayed by two standard methods e.g., castor oil-induced diarrhea and castor oil-induced gastrointestinal motility. Both, in vivo model, showed an extremely significant (P < 0.0001) dose-dependent manner percentage of inhibition in comparison to the control group. Present results suggested, A. trilobata could be a potential source for antioxidative, cytotoxic, thrombolytic, analgesic, antidiarrheal agents which require further study to identify the mechanism of A. trilobata.


Introduction
The redox homeostasis plays an essential part in maintaining health and disease prevention. The imbalance of antioxidants and reactive oxygen species (ROS) is responsible for producing oxidative stress (OS) [1]. Free radicals initiate oxidative stress resulting in DNA damage and tissue damage which caused inflammation or cell death [2]. OS is associated with the prevalence of the cardiac disease, cancers, diabetes, neurodegenerative diseases, autoimmune disorders, aging, and others. The plant derives substances such as vegetables, and dietary fruits are rich in source of antioxidant. Antioxidant suggested having a significant benefit in health by reducing oxidative stress [1]. Thrombosis is a vital physiopathology which causes several atherothrombotic diseases (e.g., myocardial or cerebral infarction). The formation of a thrombus or blood clots in the artery because of the homeostatic imbalance leads to blockage of vascular organ and while recovering causes fatal significances, myocardial, or cerebral infarction, as well as death [3]. Pain is an unsavory phenomenon that comprises sensory experiences, including time, space, force, feeling, insight, and inspiration [4]. Several analgesic agents are isolated from natural sources such as morphine, aspirin [5]. Micro-organisms like Salmonella, Escherichia coli, Vibrio cholera, and Shigella are the most regular reasons for diarrhea in developing countries [6].
Biodiversity has a significant contribution to human livelihood. As

Preparation of methanol crude extract and n-hexane fraction
The leaves and stems dried under shade and ground for ten day's period, then dried in a mechanical drier at 60-70°C. After drying, the leaves and stems were ground to a coarse powder and dissolved into methanol for 7 days. After that, the sediments filtered and dried in a water bath at 40-50°C. A concentrated filtrate like the deep green color obtained after completely evaporating the solvent, which used as methanol extract (MEAT) for the experiment. Five (5 gm) of crude extract dissolved into water and methanol. The solution then was shaken well with n-hexane. The upper portion was then separated carefully by the separating funnel. The solvent was evaporated entirely with the help of the water bath and obtained n-hexane fraction (NFAT) used for the experiment.

Standardization and quality control of the extract
Methanol (MEATL, MEATS) and n-hexane (NFATL, NFATS) extract of A. trilobata leaves and stems was standardized and under-went quality control through physicochemical evaluation of crude extract, ensuring the safety and acute toxicity study in animal model [7].

Phytochemical screening
The phytochemical analysis of the methanol extract and n-hexane fraction of A. trilobata leaves and stems carried out the standard method to evaluate the alkaloid, carbohydrate, flavonoid, terpenoids, tannins, saponins, phenols, quinones, cholesterol, proteins, steroids, starch, sterols and flavonol [18,19].

Antioxidant activity 2.7.1. DPPH free radical scavenging activity
Free radical scavenging activity of methanol extract and n-hexane fraction of A. trilobata leaves and stems, were determined by the method of Braca et al. [20]. The method based on the activity of scavenging the stable free radical 1, 1-diphenyl-2-picrylhydrazyl (DPPH). Three milliliter of 0.004% DPPH solution (4 mg DPPH in 100 mL of 95% methanol) added with the different concentrations (15.625 to 500 μg/mL) of the crude extract and the n-hexane fraction. The absorbance was taken at 517 nm after 30 min by the UV spectrophotometer.
Where, A 0 = absorbance of the control; A 1 = absorbance of the extract.
Here, lower the absorbance values, the higher will be the free radical scavenging activity [21]. The IC 50 (50% inhibitory concentration) was calculated as it indicated the effective concentration of the extract needed to scavenge 50% of the free radicals of DPPH.

Reducing power capacity
Reducing power capacity was estimated by the method described by Oyaizu (1986) [22]. One milliliter of extract taken in serially diluted concentration (31.25, 62.5, 125, 250 and 500 μg/mL) and then added 2.5 mL phosphate buffer (0.2 M; pH 6.6) and potassium ferricyanide (1% w/v), respectively and incubated at 50°C for 20 min to complete the reaction. After incubation, 2.5 mL trichloroacetic acid (10%) was added to the mixture and then centrifuged for 10 min at 3000 RPM, and 2.5 mL of the upper layer (supernatant solution) of the solution was N. Barua, et al. Biochemistry and Biophysics Reports 23 (2020) 100772 withdrawn and added 2.5 mL distilled water and 0.5 mL FeCl 3 (0.1% w/v), respectively. Then absorbance was taken at 700 nm by the UV spectrophotometer. If the absorbance of the reaction mixture increased with the increased of the concentration, then it was indicated the increased of the reducing power capacity. As a standard ascorbic acid and as a blank solution, phosphate buffer (0.2 M, pH 6.6) used.

Total phenol content
The total phenol content of the extract was measured by using Folin-Ciocalteau reagent (FCR) as an oxidizing agent by the method of Singleton et al. [23]. 2.5 mL Folion-Ciocalteau reagent (FCR) (10 times diluted with water) and 2.5 mL sodium carbonate (Na 2 CO 3 ) (20%) was mixed with 500 μg/mL extract. The mixture was made up to 10 mL by distilled water and incubated at 25°C for 20 min to complete the reaction. The absorbance taken at 765 nm. The total phenol content concentration in the extract was then determined as mg of gallic acid equivalent by the equation obtained from the graph of standard gallic acid.

Total flavonoid content
The total flavonoid content of the extract carried out by using a standard colorimetric method of Chang et al. using quercetin as standard [24,25]. In 500 μg/mL extract, 1.5 mL methanol and 100 μL aluminum chloride (AlCl 3 ) (10%) was mixed. 100 μL potassium acetate (1 M) and 2.8 mL distilled water added into the mixture. The mixture incubated at room temperature for 30 min to complete the reaction. Then absorbance was taken at 415 nm against a blank solution containing all the reagents except extract. A standard quercetin graph determined the total flavonoid content and expressed as mg of quercetin equivalent concentration.

Total flavonol content
The total flavonol content determined by adopting the method described by Kumaran and Karunakaran [26]. 500 μg/mL extract mixed with 0.5 mL AlCl 3 (5%, 20 gm/L) and 1 mL sodium acetate (50 gm/L) solution. For completing the reaction, the mixture incubated for 150 min at room temperature, and then absorbance was taken at 440 nm against a blank solution containing all the reagents except the extract. Total flavonol content calculated as mg/g of quercetin equivalent by using the equation obtained from the standard quercetin graph.

Brine shrimp lethality bioassay
The brine shrimp lethality bioassay of A. trilobata observed by using simple organism Artemia salina leach (saline arrangement shrimp eggs). In the artificial seawater (3.8% NaCl solution), the shrimp eggs hatched for 48 h for maturing the shrimp called nauplii. The cytotoxicity bioassay carried on brine shrimp nauplii following the method described by Meyer et al. [27,28]. The extract was dissolved in DMSO (50 μL in 5 mL solution) to prepare the test sample with artificial seawater (3.8% NaCl in water) to obtain the serially diluted concentrations of 31.25, 62.5, 125, 250, 500 and 1000 μg/mL. Vincristine sulfate used as a positive control as the preceding method in a serial concentration dilution 0.125, 0.25, 0.5, 1, 5, and 10 μg/mL. Ten of the living nauplii applied to each of all experimental vials and control vials. Following 24 h, all vials inspected by an amplifying glass, and the number of living nauplii in each vial was observed and recorded. % of mortality = (N 0 -N 1 /N 0 ) × 100 Where, N 0 = the number of nauplii taken; N 1 = the number of nauplii alive.

Thrombolytic activity
Thrombolytic activity test performed using the method described by Prasad et al. [29]. As a stock solution, lyophilized streptokinase vial (1500000 IU) mixed adequately with 5 mL sterile distilled water from which appropriate dilution made. Venous blood was withdrawn (5 mL) from healthy volunteers (n = 6) without the history of anticoagulant therapy or an oral contraceptive. Then distributed (0.5 mL/tube) to each six previously weight microcentrifuge tubes (sterilized) and incubated to form the clot at 37°C for 45 min. After the formation of the clot, completely removed the serum without disturbing the clot and each tube reweighed for calculating the clot weight. 100 μL extract (10 mg/mL) added to each tube having the pre-weighed clot. 100 μL streptokinase and 100 μL distilled water were added separately to the positive and negative control group. Incubation was done for 90 min at 37°C and observed clot lysis. The released fluid was removed and reweighed the tube to calculate the difference in weight after clot disruption.
% of clot lysis = (weight of clot after remove of fluid/clot weight) × 100

Acetic acid induced writhing inhibition test
The analgesic activity evaluated by the acetic acid-induced writhing test [30,31]. Before starting the test, all experimental animals were unfed for 2 h. The mice separated into ten groups (n = 5). As negative control, 1% Tween-80 solution at 10 mL/kg b.w. given orally and as a positive control (Diclofenac sodium) has been given at 25 mg/kg b.w., IP. Plant extracts (MEALT, NFALT, MEATS, and NFATS) administrate with a dose of 200 and 400 mg/kg b.w. by orally using gavage, respectively. Thirty minutes after administration, 0.7% acetic acid was injected into the mice intraperitoneally and record and count the number of writhing for 20 min.

Formalin induced paw licking test
The analgesic activity was evaluated by the formalin-induced licking test [32] with the treatment of animals of each group (n = 5), as described in the acetic acid-induced writhing test. After 30 min of the administration, 20 μL formalin (2.5% v/V) was injected into the right hind paw just under the skin of the dorsal surface by a micro-syringe having a 26-gauge needle. The licking time of the first 5 min as early phase and then 15-30 min as late phase recorded.

Castor oil-induced diarrhea
Antidiarrheal activities carried by the method Nwodo and Alumanah (1991) [33]. All experimental animals unfed for 24 h before starting the test. The mice separated into ten groups (n = 5). As negative control, 1% Tween-80 solution at 10 mL/kg b.w. given orally and as a positive control (loperamide) has been given at 5 mg/kg b.w., IP. Plant extract (MEATL, NFATL, MEATS, and NFATS) has been received orally by gavage in a dose of 200 and 400 mg/kg b.w., respectively. One hour after administration, 0.5 mL castor oil has been given orally and kept them in separate cages consist of adsorbent paper beneath. The feces were counted and observed every hour till 4 h for each mouse and replaced in every 1 h. The equation calculated the level of % inhibition of defecation: Where, A = average eradication feces number of the control group; B = average eradication feces number of the text group.

Castor oil-induced gastrointestinal motility
This gastrointestinal motility experiment was carried out by the method described by Mascolo et al. [34] with the treatment of animals of each group (n = 5) as described in the castor oil-induced diarrhea. One hour after administration of treatment group, animals treated with 1 mL charcoal meal (10% charcoal in 5% gum acacia) administered orally to each mouse. One hour later of charcoal administration, the animals were sacrificed. The distance travel charcoal meal from the pylorus to caecum was determined and presented as the total length of the intestine in percentage. The following formulae used to express the percentage of inhibition and Peristalsis index

Statistical analysis
Values were represented in Mean ± SEM (n = 5). a P < 0.05, b P < 0.01, c P < 0.001 and d P < 0.0001 indicated statistically significant in comparison to control group followed by unpaired t-test of one-way ANOVA (GraphPad Prism ver 7.0).

Qualitative phytochemical screening
The qualitative phytochemical analysis of methanolic and n-hexane extract of A. trilobata leaves and stem showed the presence of alkaloid, carbohydrate, glycosides, flavonoids, phenols, flavonol, and saponins in all four extracts. In contrast, terpenoids only present in the methanolic extract of A. trilobata leaves. The phytochemical analysis in a qualitative manner summarized in Table 1. Table 2 and Fig. 1 were summarized the scavenging activity of DPPH assay and were in the following order: ascorbic acid > NFATL > MEATL > NFATS > MEATS. The antioxidant DPPH scavenging activity of A. trilobata fractions exhibited a significant (P < 0.05) manner inhibition in compared to standard drug ascorbic acid. Among all four extracts, NFATL showed the highest scavenging activity at (89.19%) at 500 μg/mL while at the same concentration ascorbic acid exhibited 98.33% scavenging activity. IC 50 values calculated by the linear regression equation, whereas the IC 50 values of ascorbic acid and NFATL were 36.32 and 139.65 μg/mL, respectively.

Reducing power activity
In Fig. 2, the dose-response curve for reducing power activity for A.
trilobata fractions summarized (31.25-500 μg/mL). Reducing power will be increased with the increase of the concentration of the samples. The orders for reducing power activity were as followed: ascorbic acid > NFATL > NFATS > MEATL > MEATS. NFATL exhibited higher reducing power activity 0.955 at 500 μg/mL.

Total phenolic, flavonoid and flavonol contents
Quantitative analysis of antioxidant relevant phytochemicals total phenol content, total flavonoid content, and total flavonol content of A. trilobata (AT) fractions (500 μg/mL) summarized in Table 3 along with their regression equation. The orders for antioxidant relevant phytochemicals activity were as followed: NFATL > MEATL > NFATS > MEATS. NFATL showed the highest total phenol content (69.68 ± 0.67 mg GAE/g AT), total flavonoid content (53.69 ± 0.35 mg QE/g AT), and total flavonol content (153.26 ± 0.75 mg GAE/g AT) followed by MEATL, NFATS, and MEATS. Table 4 and Fig. 3 summarized the LC 50 values with the regression equation and the percentage of mortality of A. trilobata fractions, where no extract found to be toxic in comparison to positive control vincristine sulfate (2.16 μg/mL). The A. trilobata fractions exhibited moderately (MEATS = 328 μg/mL) to weakly toxic (NFATL = 616.85 μg/mL) LC 50 observed.

Thrombolytic activity
The thrombolytic activity of methanolic and n-hexane extract of A. trilobata leaves and stem summarized in Fig. 4. The MEATS showed the highest percentage of clot lysis (25.58 ± 4.76%, P < 0.0001) in comparison to negative control water (3.78 ± 0.49%), whereas the     (Table 5).

Formalin induced analgesic test
The effect of formalin-induced licking tests for analgesic activity    summarized in Table 6. The extract of methanolic and n-hexane extract of A. trilobata leaves and stem at a dose of 200 and 400 mg/kg exhibited a significant depleted manner decreased in both early and late phases. But, MEATL (400 mg/kg) showed significant (P < 0.0001) percentage of inhibition in both early and late phases (60.83% and 61.65%, respectively), which were almost similar to standard drug diclofenac Na (68.68 and 63.16%).

Castor oil-induced diarrhea in mice
The effect of castor oil-induced diarrhea in mice by A. trilobata fractions summarized in Table 7. In comparison to the negative control, the extract showed significant inhibition in both diarrhea and defecation phase in a dose-dependent manner while the MEATL 200, 400 mg/ kg showed extremely significant inhibition in defecation (P < 0.0001) and diarrhea (P < 0.001) which is higher than the standard drug loperamide.

Castor oil induced intestinal motility
The effect of castor oil-induced intestinal motility by A. trilobata fractions summarized in

Discussion
Phytochemical analysis of plant extract revealed the physiological activities as well as therapeutic activities also [35]. The phytochemical analysis of A. trilobata shows the presence of several secondary metabolites, whereas alkaloids are a particular group of secondary nitrogenous compounds which used to treat several human and animal disorder during middle age [36]. Another major phytochemical group is flavonoids, which used to treat cardiovascular diseases, cancer, and anti-inflammatory. Flavonoids usually take in dietary [37]. Presence of phenol in plants, having a contribution in physiological or biological elements of the plant [38].
Plants are an excellent source for natural antioxidants, whereas several phytochemicals have antioxidant properties. Their primary action is to ensure the protection against oxidative stress from free radicals [39,40]. Free radicals involve in several diseases like coronary infarction, atherosclerosis, neurodegenerative diseases and cancer [41]. Due to the potent antioxidant activity of polyphenolic substances (flavonoids and phenolic acids) in a biological system, are of interest [42,43]. According to literature, oxidative stress inhibited by quercetin [44] and gallic acid regulates the reactive species generation and improved a higher ratio of glutathione/oxidized glutathione [45]. Generally, the antioxidant activity was enhanced synergistically by antiradical plant phenolic substances [46]. Study reports, using of these phytochemicals substances as chemical agents (e.g., anti-inflammatory, antioxidant, anticancer), which may be useful to prevent the reactive oxygen species and antioxidant defense system [47]. In our study, all of this antioxidant relevant phytochemicals (total phenol content, total flavonoid content, and total flavonol content) of A. trilobata exhibited higher antioxidant activity in this order: NFATL > MEATL > NFATS > MEATS which also correlated with IC 50 values of DPPH scavenger. At the same time, the extract also exhibited a significant decrease in free radical scavenging assay with the correlation of concentration. The brine shrimp lethality bioassay is a safe, effective, and   Values are represented in Mean ± SEM (n = 5). a P < 0.05, b P < 0.01, c P < 0.001 and d P < 0.0001 are statistically significant in comparison to Diclofenac Na followed by unpaired t-test of one-way ANOVA (GraphPad Prism 7). MEATL: Methanolic extract of A. trilobata leaves, NFATL: n-hexane fraction of A. trilobata leaves, MEATS: Methanolic extract of A. trilobata stem and NFATS: n-hexane fraction of A. trilobata stem.
N. Barua, et al. Biochemistry and Biophysics Reports 23 (2020) 100772 economical method to determine the bioactivity of plant products [48]. The correlation between brine shrimp lethality bioassay and in vitro depletion of solid tumors in a human was introduced by the National Cancer Institute (NCI, USA). It is useful because it used as a pre-screening test for antitumor research [49]. LC 50 and toxicity are inversely proportional, whereas the higher the toxicity, the lower will be the LC 50 . The value of LC 50 over 1000 μg/mL considered to be nontoxic, ranging from 500 to 1000 μg/mL is weakly toxic, 100-500 μg/mL is moderately toxic while less than 100 μg/mL is considered as highly toxic [50]. In our study, no A. trilobata extracts found to be toxic in comparison to Vincristine sulfate, while a moderately (MEATS = 328 μg/mL) to weakly toxic (NFATL = 616.85 μg/mL) LC 50 observed. The observed cytotoxic activity may be present of alkaloid phytochemical in the extract [51], which required further study to evaluate the cytotoxic compounds.
The available drugs in the market for thrombolytic, especially streptokinase, convert plasminogen to plasmin and increased lysis of the clot. The researcher revealed that flavonoids (plant metabolites) affect embolus and cardiovascular disease by interfering with platelet activation which is a risk factor for a cardiac disorder [52,53]. Our study suggests that the MEATS and MEATL could be a potential source to inhibit the clot formation. The current finding of A. trilobata through clot lysis in blood supports the earlier study on different plant species of the Passifloraceae family [54].
Nociception triggered or activated by mediators, which cause inflammation, edema, and diapedesis or leukocyte eruption [55]. Acetic acid-induced in mice is a behavioral analgesia mediated observation where IP administrations of acetic acid produce increase prostaglandin in the fluid of peritoneal [56]. By inhibiting the prostaglandin synthesis by any chemical substances which lower the number of writhing mentioned as analgesia. In contrast, central and peripheral antinociceptive pain is a biphasic method for formalin-induced licking test. Values are represented in Mean ± SEM (n = 5). a P < 0.05, b P < 0.01, c P < 0.001 and d P < 0.0001 are statistically significant in comparison to Diclofenac Na followed by unpaired t-test of one-way ANOVA (GraphPad Prism 7). MEATL: Methanolic extract of A. trilobata leaves, NFATL: n-hexane fraction of A. trilobata leaves, MEATS: Methanolic extract of A. trilobata stem and NFATS: n-hexane fraction of A. trilobata stem. Values are represented in Mean ± SEM (n = 5). b P < 0.01, c P < 0.001 and d P < 0.0001 are statistically significant in comparison to Tween-80 (Control) followed by unpaired t-test of one-way ANOVA (GraphPad Prism 7). MEATL: Methanolic extract of A. trilobata leaves, NFATL: n-hexane fraction of A. trilobata leaves, MEATS: Methanolic extract of A. trilobata stem and NFATS: n-hexane fraction of A. trilobata stem. The early phase and late phase is neurogenic and inflammatory pain response, respectively. Narcotics inhibit both phases while the nonsteroidal anti-inflammatory drug (NSAID) inhibits the late phase [57].
In an acetic acid test, the extract of A. trilobata showed a significant dose-dependent manner percentage of inhibition in the nociceptive nerve, whereas the methanolic and n-hexane fraction of A. trilobata leaves showed the highest percentage of inhibition. Similarly, the administration of methanolic and n-hexane fraction of A. trilobata leaves and stem showed significant inhibition in both phases in a depleted manner, whereas the MEATL (400 mg/kg) showed the highest percentage of inhibition. The result showed significant inhibition in analgesic activity might be for the presence of alkaloids [58]. At the same time, the saponins and flavonoids phytochemicals are responsible for the anti-inflammatory properties of medicinal plants [59,60].The analgesic activity might also show because of the traditional used in pain sensation [15,16].
In antidiarrheal screening, castor oil cause water and electrolyte penetrability changes in the intestinal mucosal layers, bringing about liquid and watery luminal content that rapidly eliminate by intestines [61]. Ricinoleic acid is a natural laxative which is the active metabolite of castor oil, used in evaluating castor oil-induced diarrheal activity. Castor oil act on the small intestine to change the action of smooth muscle GI [62]. In our study, both models for antidiarrheal showed extremely significant inhibition of diarrhea. In contrast, the castor oilinduced diarrhea model exhibited extremely significant inhibition in defecation (P < 0.0001) and diarrhea (P < 0.001) by MEATL (200, 400 mg/kg), which is higher than the standard drug loperamide. Though the extremely significant activity in the castor oil-induced diarrhea model, it did not possess similar activity in castor oil-induced intestinal motility test. In contrast, the NFATL showed the maximum inhibition of intestinal motility. But, the finding of both models suggesting that the A. trilobata could be a potential source for diarrheal treatment, which proven the traditional used in stomach trouble [15,16].

Conclusion
The study reported that A. trilobata could be a potential source for antioxidant, cytotoxic, thrombolytic, analgesic, antidiarrheal activity due to the presence of secondary metabolites (e.g., alkaloid, flavonoid, phenol). Furthermore, studies highly recommended in identifying the mechanism of A. trilobata because there are no scientific reports related to biological activity.

Ethical approval
The study approved by the Institutional Animal Ethical Committee, Department of Pharmacy, International Islamic University Chittagong, Bangladesh according to governmental guidelines under the reference of Pharm/PND/150/20-2019.

Consent for publication
All authors have agreed to publish all materials belongs to this article.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Funding
This work is conducted with the individual funding of all authors.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.