# Medicinal Attributes of Garuga pinnata Roxb.: A Systematic Exploration of Its Analgesic, Antidiarrheal, Hypoglycemic, and Cytotoxic Activities

Mohi Uddin, Mohaiminullah Sarker, Md. Jahirul Islam Mamun, Jannatul

Mysha Binte Quader, Md. Abdul Alim, Jannatul Naima Meem

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Mohi Uddin, Mohaiminullah Sarker, Md. Jahirul Islam Mamun, Jannatul Mysha Binte Quader, Md. Abdul Alim, et al.. Medicinal Attributes of Garuga pinnata Roxb.: A Systematic Exploration of Its Analgesic, Antidiarrheal, Hypoglycemic, and Cytotoxic Activities. Asian Journal of Research in Botany, 2025, 8 (2), pp.537-554. 10.9734/ajrib/2025/v8i2285. hal-05310176

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# Medicinal Attributes of *Garuga pinnata* Roxb.: A Systematic Exploration of Its Analgesic, Antidiarrheal, Hypoglycemic, and Cytotoxic Activities

Mohi Uddin a*, Mohaiminullah Sarker a,
Md. Jahirul Islam Mamun a,
Jannatul Mysha Binte Quader a,b,
Md. Abdul Alim a and Jannatul Naima Meem a

## Authors' contributions

This work was carried out in collaboration among all authors. Authors MU and MS designed the experiments and conception. Author MS conducted the research work, data interpretation, and analysis. Authors MJIM, JMBQ, MAA and JNM wrote the manuscript. Author MU discussed the results, and authors JMBQ and MU edited and approved the final manuscript. All authors read and approved the final manuscript.

## Article Information

DOI: https://doi.org/10.9734/ajrib/2025/v8i2285

Open Peer Review History:

This journal follows the Advanced Open Peer Review policy. Identity of the Reviewers, Editor(s) and additional Reviewers, peer review comments, different versions of the manuscript, comments of the editors, etc are available here: https://pr.sdiarticle5.com/review-history/143688

Original Research Article

Received: 05/07/2025

Published: 07/10/2025

<footnote>a Department of Pharmacy, Faculty of Biological Sciences, University of Chittagong, Chittagong-4331, Bangladesh.</footnote>

<footnote>b Department of Pharmacy, Faculty of Science and Engineering, BGC Trust University Bangladesh, Kanchannagar-4381, Bangladesh.</footnote>

<footnote>*Corresponding author: E-mail: mohiuddinahmed.du@gmail.com;</footnote>

<footnote>**Cite as:** Mohi Uddin, Mohaiminullah Sarker, Md. Jahirul Islam Mamun, Jannatul Mysha Binte Quader, Md. Abdul Alim, and Jannatul Naima Meem. 2025. "Medicinal Attributes of Garuga Pinnata Roxb.: A Systematic Exploration of Its Analgesic, Antidiarrheal, Hypoglycemic, and Cytotoxic Activities". Asian Journal of Research in Botany 8 (2):537-554. https://doi.org/10.9734/ajrib/2025/v8i2285.</footnote>
ABSTRACT

This study strived to investigate the phytochemical components and therapeutic potentials of *Garuga pinnata* Roxb. (Burseraceae) leaves, a prominent source of natural product-derived medicine, including analgesic, antidiarrheal, hypoglycemic, and cytotoxic properties. The methanolic extract of *G. pinnata* leaves (MEGP) and its N-hexane (NHGP) and ethyl acetate (EAGP) fractions were evaluated upon the completion of phytochemical screening using both *in vivo* (Swiss albino mice) and *in vitro* approaches. The analgesic activity was conducted through the acetic acid-induced writhing method and the tail immersion method, while the antidiarrheal activity was evaluated using the castor oil-induced diarrheal model. The hypoglycemic effect was assessed through an oral glucose tolerance test (OGTT), whereas the brine shrimp lethality assay (BSLA) was used to determine the cytotoxic potential of the extract. Phytochemical screening confirmed the presence of phenols, flavonoids, alkaloids, and tannins. In the writhing test, MEGP at 400 mg/kg exhibited the highest analgesic effect (p<0.001). Similarly, in the tail immersion test, MEGP at 400 mg/kg showed maximum response time (7.25 ± 0.11 s, p<0.001), indicating significant central analgesic activity. MEGP demonstrated higher inhibition of defecation and diarrhea compared to other fractions, highlighting its efficacy in reducing diarrheal symptoms. MEGP at 400 mg/kg significantly reduced blood glucose levels (4.6 ± 0.27 mmol/L, p<0.001), suggesting potent hypoglycemic activity. MEGP exhibited strong cytotoxic effects with an LC₅₀ value of 52.58 µg/mL, compared to the standard vincristine sulfate (LC₅₀ = 1.71 µg/mL). *G. pinnata* shows promise as a rich source of bioactive compounds for the treatment of pain, diarrhea, diabetes, and cancer. Further studies are required to differentiate and characterize the active constituents responsible for these pharmacological activities.

Keywords: *Garuga pinnata*; analgesic; antidiarrheal; hypoglycemic; cytotoxic; tail immersion.

1. INTRODUCTION

Natural products are gaining more attention due
to their diverse bioactive constituents and
comparatively low toxicity all around the world
(Hasan et al., 2025). The use of natural
substances in therapy has a long history,
primarily because of their ability to manage
complex medical conditions while producing
relatively few side effects and remaining cost-
effective. Medicinal plants, in particular, derive
their therapeutic value from the diverse range of
chemical compounds that occur naturally within
them. Over the years, scientific inquiry has
allowed researchers to isolate and describe
numerous phytochemicals, many of which
demonstrate preventive benefits and exert
diverse physiological effects. In recent decades,
rapid advances in technology and analytical
approaches—most notably metabolomics and
genome mining—have provided powerful tools
for exploring these bioactive constituents. Such
progress has not only deepened our
understanding of the pharmacological potential of
medicinal plants but also accelerated the
identification of novel compounds with promising
therapeutic applications (Quader et al., 2024).

Pain is a complex and variable phenomenon,
commonly treated with analgesics, such as

nonsteroidal anti-inflammatory drugs (NSAIDs),
which often lead to adverse effects like
gastrointestinal discomfort, ulceration, and
bleeding, while opiates carry risks of tolerance
and dependency (Mohammad, Islam, et al.,
2025).

"Diarrhea is defined as the passage of three or more loose stools and is characterized by increased gastrointestinal motility, excessive secretion, and reduced absorption of fluids and electrolytes" (Mekonnen et al., 2018). It remains a significant cause of morbidity and mortality, particularly among children and young animals in developing countries. Medicinal plants are considered a promising source of antidiarrheal drugs" (Almeida et al., 1995; Maikere-Faniyo et al., 1989), prompting international organizations like the WHO to encourage research into traditional medicinal practices for the treatment and prevention of diarrheal diseases (Atta & Mouneir, 2004). Currently available drugs often come with adverse effects, contraindications, and challenges such as antibiotic resistance, limiting their efficacy (Administration, 2016; Alam & Bhatnagar, 2006). The high prevalence of diarrhea in developing countries, combined with limited access to healthcare and the drawbacks of existing treatments, highlights the potential of traditional medicines as effective and accessible alternatives for managing this condition.
Hyperglycemia is another health problem related to diabetes mellitus, which can be attributed to an immediate or long-term insulin deficit, along with cellular insulin resistance. Insulin and sulfonylureas effectively alleviate acute symptoms but fail to cure the disease or prevent its chronic complications (Larner & Haynes Jr, 1985). "This limitation highlights the need for continued research into alternative hypoglycemic agents, particularly those derived from plants, because plants have been a cornerstone of traditional medicine, used to treat various ailments in both humans and animals from the dawn of human civilization" (Akhtar, 1992).

Cancer remains one of the leading causes of death globally, prompting significant efforts to develop effective strategies to combat this disease. Chemotherapy is a cornerstone of modern cancer treatment, with numerous clinically available drugs used to manage leukemia, lymphoma, and solid tumors (Behzad et al., 2014). Notably, many plant-derived compounds have proven highly successful in cancer chemotherapy, underscoring the critical role of plants as a source of novel anticancer agents. Researchers have highlighted the significance of natural products, with over 50% of approved chemotherapeutic agents being derived from or inspired by natural compounds (Cragg & Newman, 2000). This emphasizes the continued importance of exploring plants for the discovery of new and effective anticancer therapies.

"Garuga pinnata" Roxb. (Burseraceae), locally known as Nilbhadi, is widely distributed across India, often found in mixed deciduous forests as a sporadic associate of teak and sal trees" (Shirwaikar et al., 2006). "Traditionally, its leaves are used to treat conjunctivitis-related opacities, while its fruits are employed as a stomachic and cooling agent" (Nadkarni, 2007). "The leaf juice, when mixed with honey, is utilized for asthma treatment" (Kirtikar & Basu, 1991). Scientific studies have demonstrated that the aqueous extract of its stem bark inhibits lipid peroxidation (Shirwaikar et al., 2007), and its methanol extract exhibits potent free radical scavenging activity in vitro (Thupurani et al., 2012). "Additionally, the methanol extract of the stem bark has shown promising anticancer effects on human breast cancer cell lines, such as MCF-7 and MDA-MB-231" (Thupurani et al., 2013). In light of these pharmacological properties, the present investigation aims to elucidate the analgesic,

antidiarrheal, hypoglycemic, and cytotoxic activities of *G. pinnata* leaves through *in vivo* and *in vitro* approaches.

## 2. MATERIALS AND METHODS

### 2.1 Drugs and Chemicals

We bought analytical-grade methanol, chloroform, and colchicine from Merck, Germany. Glibenclamide 75 mg (BEXIMCO Bangladesh Ltd., Dhaka, Bangladesh), diclofenac sodium 100 mg SQUARE Pharmaceuticals Ltd., Dhaka, Bangladesh), and loperamide 10 mg (MSL tablet, Uni Health Pharmaceutical Ltd., Dhaka, Bangladesh) were among the standard medications gathered from different manufacturers. For specific assays, all commercially available standard medications were utilized as stock solutions without additional purification.

### 2.2 Collection and Identification of the Plant

The leaves of *Garuga pinnata* were gathered from the Botanical Garden of the University of Chittagong, Chattogram, Bangladesh, in October 2024. The plant was authenticated as *Garuga pinnata* Roxb. by Dr. Shaikh Bokhtear Uddin, Taxonomist and Professor, Department of Botany, University of Chittagong.

### 2.3 Preparation of Extract

Following the drying process of the leaves, a coarse powder was created using a grinding machine (Xiangrong, China). Before additional examination, the powder was kept in a dark atmosphere at a temperature of 30 °C ± 2 °C and a relative humidity of 70 ± 5%. Two hundred grams of powdered *G. pinnata* were measured using an analytical balance (AS 202.R2 Plus Radwag). A Soxhlet apparatus (SE-250~2000, Guangdong, China, Pyrex, Glass) was used to extract the powder rapidly using 1000 milliliters of methanol. At temperatures kept below 50 °C, the solvent was evaporated using a collection, filtering, and rotary evaporation method (Wincom, 52A, Guangdong, China). A sticky concentration consistency was produced after the solvent evaporated. To get the required amounts, it was repeated multiple times, and the extracts were mixed. Before the test, the *G. pinnata* leaf extract was kept in a glass vial at 4°C (Hossain et al., 2025).
## 2.4 Solvent-solvent Partition

The dried methanolic crude extract was suspended in distilled water and then subjected to solvent-solvent partitioning using a modified Kupchan technique (Kupchan & Tsou, 1973). N-hexane and ethyl acetate were successively fractionated in separatory funnels to yield the relevant solvent fractions. Each solvent fraction was dried under low pressure in a rotary evaporator and stored at 4 °C for further pharmacological analysis.

## 2.5 Phytochemical Screening

The sample was first screened for the presence of secondary metabolites using previously published techniques with minor adjustments (Bhandari et al., 2021) (Nahar et al., 2025).

## 2.6 Experimental Animals

The Bangladesh Council of Scientific and Industrial Research (BCSIR), Chattogram and Rajshahi, provided us with healthy Swiss albino mice of both sexes, weighing 20–25 g and 6–7 weeks, for our study. For two weeks before the trials, the mice were kept in plastic cages with a controlled temperature of 25 ± 1 °C, a humidity of 55–65%, and a 12-hour light/dark cycle (Mohammad, Mamun, et al., 2025) (Mohammad, Chowdhury, et al., 2025). They were also given water and food prepared by BCSIR. They were made to feel less uncomfortable. The research methodology was conducted in accordance with the guidelines of the US National Institutes of Health for animal care and use. It was approved by the University of Chittagong's Faculty of Biological Sciences' Ethical Committee for Animal Use (AERB-FBSCU-/25/6). Mice were euthanized using diethyl ether anesthesia at the conclusion of the experiment. All experimental procedures were conducted in accordance with the ARRIVE guidelines to ensure ethical and standardized reporting of animal research.

## 2.7 Acute Oral Toxicity

Under OECD rules for chemical testing, Test No. 423 (Oecd, 2001), an acute oral toxicity study was conducted. In the toxicity research, five rats were given oral gavages of GP at single doses of 1000, 2000, or 4000 mg per kilogram of body weight. Food was stopped for three to four hours

after the dose was administered. Throughout the first 30 minutes following medication and at regular intervals for the first 24 hours, each animal was attentively monitored. To document any delayed toxicity, they were monitored for a total of 14 days (Jagannath et al., 2025). Autonomic and central nervous system (CNS) function, respiratory and circulatory rates, mucosal membranes, skin, fur, and eyes were all altered. The effective therapeutic dose was determined using the median lethal dose (LD50 > 2.0 g/kg) (Tareq et al., 2020).

## 2.8 Analgesic Activity

### 2.8.1 Acetic acid-induced writhing test

A common technique for assessing G. pinnata's analgesic potential was the acetic acid-induced writhing method (Hossen et al., 2021). Two doses of the test extracts (200 and 400 mg/kg body weight), vehicle (10 mL/kg body weight), and diclofenac sodium (10 mg/kg body weight) were administered orally 40 minutes before the intraperitoneal injection of 0.7% acetic acid following an overnight fast. Each animal's total number of writhings was recorded for 15 minutes, five minutes after the acetic acid was administered. The percentage inhibition of writhing was calculated as follows (Ahsan et al., 2025):

$$ \% \text{Inhibition} = \frac{A - B}{A} \times 100 $$

Here, A = Average writhes in the control group, B = Average writhes in the test group.

### 2.8.2 Tail immersion test

The tail immersion method, which was devised and described by Gawel et al. (Gawel et al., 2018), was used to evaluate the central analgesic property. This approach is predicated on the finding that medications with morphine-like effects can prolong the mice's tail's abandonment from warm water. One to two centimeters of the rodents' tail was immersed in hot water that was kept at 54 ± 0.5 °C after the animal had been treated with diclofenac sodium or extracts. The latency duration of the tail-withdrawal reaction was measured at 30, 60, 90, and 120 minutes following the administration of the medication and extracts. To prevent harm to the tail tissue, the latency period was limited to 20 seconds.
## 2.9 Antidiarrheal activity

### 2.9.1 Castor oil-induced diarrhea

This test was performed based on the method described by Awouters et al. (Awouters et al., 1978). Twenty mice were fasted for 18 hours and divided into four groups, each containing five animals. The first group received distilled water (10 ml/kg) as the negative control, while the second group received loperamide (3 mg/kg) as the positive control. Groups 3 to 8 were administered 200 and 400 mg/kg of the extract, respectively. After one hour, all animals were orally given 0.5 ml of castor oil and placed in separate metabolic cages. The severity of diarrhea was assessed over 4 hours by counting the total number of feces (dry and wet). The mean total fecal output for the negative control group was considered 100%, and the percent inhibition of total defecation and diarrhea was calculated using the following formulas:

$$ \% inhibition of defecation = \frac{A - B}{A} \times 100 $$

Here, A = Total number of feces in the negative control,

B = Total number of feces in the treated group

$$ \% inhibition of diarrhea = \frac{A - B}{A} \times 100 $$

Here, A = Total number of diarrheal feces in the negative control,

B = Total number of diarrheal feces in the treated group.

## 2.10 Hypoglycemic Activity

### 2.10.1 Glucose tolerance test

The efficacy of the leaf extracts that showed hypoglycemia potential in the screening tests was assessed using the Oral Glucose Tolerance Test (OGTT). Mice that had fasted overnight were given extracts (MLGP/NHGP/EAGP) at concentrations of 200 mg/kg or 400 mg/kg; the control group received NaCl 0.9%. Blood glucose levels were assessed before and 120 minutes following the administration of oral extracts and intraperitoneal injections, respectively. Following this time point, 3 g/kg of oral glucose was given, and the OGTT was used to test blood glucose levels at 0, 30, 60, and 120 minutes (Arya et al., 2012).

## 2.11 Cytotoxic activity

### 2.11.1 Brine shrimp lethality assay

A brine shrimp lethality assay was conducted to evaluate the cytotoxic potential of various plant extracts. To simulate saltwater, 38 g of NaCl was dissolved in 1000 mL of distilled water, with NaOH added to maintain a consistent pH (Kooti et al., 2017). Brine shrimp eggs were hatched in this synthetic saltwater to obtain nauplii. Dimethyl sulfoxide (DMSO) was used to prepare test samples at varying concentrations (6.25 µg/mL to 400 µg/mL). Vincristine sulphate served as the reference standard at the concentration range (0.3125 µg/mL to 20 µg/mL), while DMSO acted as the negative control. Nauplii were visually counted at room temperature (25°C) and transferred to vials containing 5 mL of simulated saltwater. Samples at different concentrations were added to the pre-marked vials using a micropipette. After 24 hours, the surviving nauplii were recorded to assess cytotoxicity (Mohammad, Rasel, et al., 2025).

$$ \% Mortality = \frac{A - B}{A} \times 100 $$

Here, A = Number of nauplii taken,

B = Number of nauplii alive

## 2.12 Statistical Analysis

The mean ± standard error of the mean (SEM) is how the data are displayed. A one-way ANOVA following the Dunnett t-test was used for statistical analysis. At the *p < 0.05, **p < 0.01, and ***p<0.001 levels, differences are considered statistically significant. MS Excel 2024 and GraphPad Prism 8.0.1 software were used to process the results.

# 3. RESULTS

## 3.1 Phytochemical Screening

The phytochemical results of the methanolic leaf extract of *G. pinnata* are shown in Table 1.

## 3.2 Acute Oral Toxicity

The methanolic leaf extract of *G. pinnata* was determined to be safe in the oral acute toxicity test with a limit dose of 2000 mg/kg body weight since no mortality or notable toxicity symptoms were observed in the 14-day observation of the test mice. This outcome demonstrates that the plant extract's LD₅₀ is higher than 2000 mg/kg.
<table><caption>Table 1. Result of phytochemical screening of the methanolic leaf extract of *G. pinnata*</caption><thead><tr><th>SL No.</th><th>Phytochemicals</th><th>Methanolic extract of G. pinnata leaves</th></tr></thead><tbody><tr><td>1</td><td>Alkaloids</td><td>+</td></tr><tr><td>2</td><td>Tannins</td><td>+</td></tr><tr><td>3</td><td>Flavonoids</td><td>+</td></tr><tr><td>4</td><td>Saponins</td><td>+</td></tr><tr><td>5</td><td>Glycosides</td><td>+</td></tr><tr><td>6</td><td>Cardiac glycosides</td><td>-</td></tr><tr><td>7</td><td>Carbohydrates</td><td>-</td></tr><tr><td>8</td><td>Proteins</td><td>-</td></tr><tr><td>9</td><td>Terpenoids</td><td>+</td></tr><tr><td>10</td><td>Steroids</td><td>+</td></tr><tr><td>11</td><td>Phenols</td><td>+</td></tr></tbody></table>

Note: (+) = Presence of phytochemicals; (-) = Absence of phytochemicals.

## 3.3 Analgesic Activity

### 3.3.1 Acetic acid-induced writhing method

The acetic acid-induced writhing inhibition method was used to screen for analgesic efficacy in the methanolic leaf extract of *G. pinnata* and its various solvent-soluble fractions, and the extracts were administered at 200 and 400 mg/kg body weight to conduct the test. Table 2 displays the outcome. Compared to the control, which was equivalent to the standard medication diclofenac sodium (59.70% inhibition, p < 0.001), MLGP caused a significant (p < 0.001) reduction in the number of writhing in a dose-dependent manner, with 43.28 % and 31.34 % of inhibition at the dose of 400 mg/kg and 200 mg/kg body weight, respectively. At 400 mg/kg and 200 mg/kg body weight, respectively, the soluble fractions of ethyl

acetate and n-hexane demonstrated dose-dependent moderate to mild antinociceptive efficacy (Fig. 1).

### 3.3.2 Tail immersion method

The test involved administering extracts at doses of 200 mg/kg and 400 mg/kg body weight, with the tail withdrawal reflex time showing nearly similar increases with higher sample doses compared to the standard drug (Fig. 2). A highly significant effect (p < 0.001) was observed following treatment with MLGP at 400 mg/kg after 90 minutes, yielding a mean reaction time of 7.25 ± 0.11. Similarly, NHGP and EAGP administered at the same dose of 400 mg/kg also showed notable effects at 90 minutes, with reaction times of 5.07 ± 0.19 and 5.33 ± 0.11, respectively (Table 3).

<figure><img src="image_2.png" /><figcaption>Fig. 1. Evaluation of Analgesic Activity of *G. pinnata* through the Acetic Acid-Induced Writhing Method</figcaption></figure>
<table><caption>Table 2. Evaluation of Analgesic activity of methanolic leaf extract and its different fractions of G. pinnata</caption><thead><tr><th>Treatment</th><th>Dose (mg/kg)</th><th>Number of Writhing (Mean ± SEM)</th><th>% Inhibition of Writhing</th></tr></thead><tbody><tr><td>Control</td><td>10 mL/kg</td><td>40.20 ± 1.43</td><td>-</td></tr><tr><td>Diclofenac Na</td><td>10</td><td>16.20 ± 0.68***</td><td>59.70</td></tr><tr><td>MLGP</td><td>200</td><td>27.60 ± 2.14***</td><td>31.34</td></tr><tr><td>MLGP</td><td>400</td><td>22.80 ± 1.71***</td><td>43.28</td></tr><tr><td>NHGP</td><td>200</td><td>36.00 ± 1.70</td><td>10.45</td></tr><tr><td>NHGP</td><td>400</td><td>32.20 ± 1.88*</td><td>19.90</td></tr><tr><td>EAGP</td><td>200</td><td>31.80 ± 2.42**</td><td>20.90</td></tr><tr><td>EAGP</td><td>400</td><td>29.00 ± 1.22***</td><td>27.86</td></tr></tbody></table>

Note: Values are expressed as Mean ± SEM; n=5. One-way ANOVA followed by Dunnett's t-test. ***p < 0.001, **p < 0.01, *p < 0.05 is statistically significant with compared to control. MLGP = Methanolic leaves extract of G. pinnata; NHGP = N-hexane soluble fraction of G. pinnata leaves; EAGP = Ethyl acetate soluble fraction of G. pinnata leaves.

<table><caption>Table 3. Evaluation of Analgesic activity of methanolic leaf extract and its different fractions of G. pinnata</caption><thead><tr><th rowspan="2">Treatment</th><th rowspan="2">Dose (mg/kg)</th><th colspan="5">Response time (sec)</th></tr><tr><th>0 min</th><th>30 min</th><th>60 min</th><th>90 min</th><th>120 min</th></tr></thead><tbody><tr><td>Control</td><td>10 mL/kg</td><td>2.76 ± 0.11</td><td>3.02 ± 0.05</td><td>3.29 ± 0.07</td><td>3.77 ± 0.07</td><td>3.96 ± 0.09</td></tr><tr><td>Morphine</td><td>10</td><td>2.63 ± 0.06</td><td>7.04 ± 0.11***</td><td>8.61 ± 0.12***</td><td>8.21 ± 0.09***</td><td>7.58 ± 0.12***</td></tr><tr><td>MLGP</td><td>200</td><td>2.72 ± 0.16</td><td>3.66 ± 0.14**</td><td>4.44 ± 0.17***</td><td>5.15 ± 0.14***</td><td>5.01 ± 0.10***</td></tr><tr><td>MLGP</td><td>400</td><td>2.51 ± 0.14</td><td>4.64 ± 0.15***</td><td>5.95 ± 0.26***</td><td>7.25 ± 0.11***</td><td>6.38 ± 0.29***</td></tr><tr><td>NHGP</td><td>200</td><td>2.79 ± 0.11</td><td>3.26 ± 0.14</td><td>3.65 ± 0.09</td><td>4.19 ± 0.13</td><td>4.2 ± 0.14</td></tr><tr><td>NHGP</td><td>400</td><td>2.65 ± 0.11</td><td>3.46 ± 0.17</td><td>4.24 ± 0.14***</td><td>5.07 ± 0.19***</td><td>4.97 ± 0.13***</td></tr><tr><td>EAGP</td><td>200</td><td>2.68 ± 0.10</td><td>3.26 ± 0.14</td><td>3.85 ± 0.13</td><td>4.32 ± 0.10*</td><td>4.2 ± 0.14</td></tr><tr><td>EAGP</td><td>400</td><td>2.48 ± 0.10</td><td>4.06 ± 0.12***</td><td>5.04 ± 0.11***</td><td>5.33 ± 0.11***</td><td>5.13 ± 0.08***</td></tr></tbody></table>

Note: Values are expressed as Mean ± SEM; n=5. One-way ANOVA followed by Dunnett's t-test. ***p < 0.001, **p < 0.01, *p < 0.05 is statistically significant with compared to control. MLGP = Methanolic leaves extract of G. pinnata; NHGP = N-hexane soluble fraction of G. pinnata leaves; EAGP = Ethyl acetate soluble fraction of G. pinnata leaves.
<figure><img src="image_3.png" /><figcaption>Fig. 2. Evaluation of Analgesic Activity of *G. pinnata* through the Tail Immersion Method</figcaption></figure>

**3.4 Antidiarrheal Activity**

**3.4.1 Effects on castor oil-induced diarrhea**

Table 4 demonstrates the antidiarrheal activity of the methanolic leaf extract of *G. pinnata* and its different solvent fractions at two dose levels, observed over 4 hours after castor oil

administration. When compared to the negative control, only the higher dose of the MLGP extract significantly decreased both the total number of faecal droppings (p < 0.001) and the number of diarrheal stools (p < 0.01), resulting in a 67.82% inhibition of defecation and a 62.05% reduction in diarrhoea (Fig. 3).

<figure><img src="image_4.png" /><figcaption>Fig. 3. Evaluation of Antidiarrheal Activity of *G. pinnata* through the Castor Oil-Induced Diarrheal Test</figcaption></figure>
## 3.5 Hypoglycemic Activity

### 3.5.1 Oral glucose tolerance test (OGTT)

Using an oral glucose tolerance test, the methanolic extract of *G. pinnata* and its multiple fractions at doses of 400 mg/kg and 200 mg/kg body weight were screened for hypoglycemic action. Table 5 displays the outcome. Both the 400 mg/kg and 200 mg/kg dosages of the extract MEGP were shown to significantly (p < 0.001) lower blood glucose levels at 60 minutes when compared to the control; however, the 400 mg/kg dose of MLGP exhibited greater hypoglycemic activity (4.6 ± 0.27), which was closer to that of the standard glibenclamide (3.52 ± 0.12). The other

fractions showed moderate hypoglycemic activity (Fig. 4).

## 3.6 Cytotoxic Activity

### 3.6.1 Brine shrimp lethality test

The cytotoxic activity of *G. pinnata*'s crude methanolic extract and its various soluble fractions against brine shrimp nauplii was examined. Comparing the MLGP extract to the standard medication (1.71 µg/mL), the LC₅₀ value was 52.58 µg/mL, indicating significant cytotoxicity. The NHGP and EAGP fractions had LC₅₀ values of 111.39 µg/mL and 129.28 µg/mL, respectively, which showed modest cytotoxicity when compared to the standard. Figs. 5 and 6 exhibit the findings.

<figure><img src="image_5.png" /><figcaption>Fig. 4. Evaluation of Hypoglycemic Activity of *G. pinnata* through the Oral Glucose Tolerance Test</figcaption></figure>

<table><caption>Table 4. Effects of methanolic leaf extract and its different fractions of *G. pinnata* on castor oil-induced diarrhoea</caption><thead><tr><th>Treatment (mg/kg)</th><th>Total number of feces</th><th>Inhibition of defecation (%)</th><th>Total number of diarrheal feces</th><th>Inhibition of diarrhea (%)</th></tr></thead><tbody><tr><td>Control</td><td>17.4 ± 0.93</td><td>-</td><td>9.67 ± 0.88</td><td>-</td></tr><tr><td>Loperamide (5 mg/kg)</td><td>5.2 ± 0.37***</td><td>70.11</td><td>2.33 ± 0.33**</td><td>75.90</td></tr><tr><td>MLGP 200</td><td>6.2 ± 0.58***</td><td>64.37</td><td>4.67 ± 0.33*</td><td>51.71</td></tr><tr><td>MLGP 400</td><td>5.6 ± 0.51***</td><td>67.82</td><td>3.67 ± 0.88**</td><td>62.05</td></tr><tr><td>NHGP 200</td><td>15.2 ± 0.66</td><td>12.64</td><td>9.33 ± 1.33</td><td>3.52</td></tr><tr><td>NHGP 400</td><td>12.2 ± 0.66***</td><td>29.89</td><td>8.67 ± 1.45</td><td>10.34</td></tr><tr><td>EAGP 200</td><td>9.2 ± 0.58***</td><td>47.13</td><td>8.33 ± 1.20</td><td>13.86</td></tr><tr><td>EAGP 400</td><td>7.4 ± 0.51***</td><td>57.47</td><td>7.67 ± 0.67</td><td>20.68</td></tr></tbody></table>

Note: Values are expressed as Mean ± SEM; n=5. One-way ANOVA followed by Dunnett's t test. ***p < 0.001, **p < 0.01, *p < 0.05 is statistically significant with compared to control. MLGP = Methanolic leaves extract of *G. pinnata*; NHGP = N-hexane soluble fraction of *G. pinnata* leaves; EAGP = Ethyl acetate soluble fraction of *G. pinnata* leaves.
A

<figure><img src="image_6.png" /></figure>

B

<figure><img src="image_7.png" /></figure>
C

<figure><img src="image_8.png" /></figure>

D

<figure><img src="image_9.png" /><figcaption>Fig. 5. Plot of percentage of mortality and regression line of A) Vincristine sulfate, B) MLGP, C) NHGP, and D) EAGP. MLGP = Methanolic leaves extract of *G. pinnata*; NHGP = N-hexane soluble fraction of *G. pinnata* leaves; EAGP = Ethyl acetate soluble fraction of *G. pinnata* leaves</figcaption></figure>
<table><caption>Table 5. Hypoglycemic activity of methanolic leaf extract of G. pinnata</caption><thead><tr><th rowspan="2">Treatment (mg/kg)</th><th colspan="4">Blood Glucose Level (mmol/L)</th></tr><tr><th>0 min</th><th>30 min</th><th>60 min</th><th>120 min</th></tr></thead><tbody><tr><td>Control</td><td>4.4 ± 0.07</td><td>14.42 ± 0.10</td><td>7.76 ± 0.25</td><td>6.18 ± 0.12</td></tr><tr><td>Glibenclamide (10 mg/kg)</td><td>4.26 ± 0.12</td><td>10.66 ± 0.29***</td><td>3.98 ± 0.18***</td><td>3.52 ± 0.12***</td></tr><tr><td>MLGP 200</td><td>4.18 ± 0.10</td><td>12.5 ± 0.41**</td><td>5.3 ± 0.19***</td><td>4.94 ± 0.27**</td></tr><tr><td>MLGP 400</td><td>4.16 ± 0.12</td><td>11.98 ± 0.51**</td><td>4.7 ± 0.16***</td><td>4.8 ± 0.27***</td></tr><tr><td>NHGP 200</td><td>4.21 ± 0.16</td><td>12.34 ± 0.66*</td><td>6.67 ± 0.36</td><td>5.17 ± 0.44</td></tr><tr><td>NHGP 400</td><td>4.14 ± 0.12</td><td>13.12 ± 0.44*</td><td>5.44 ± 0.58**</td><td>4.98 ± 0.66**</td></tr><tr><td>EAGP 200</td><td>4.23 ± 0.24</td><td>10.67 ± 0.26***</td><td>6.13 ± 0.88</td><td>5.46 ± 0.58</td></tr><tr><td>EAGP 400</td><td>4.20 ± 0.10</td><td>11.66 ± 0.24**</td><td>6.05 ± 0.24*</td><td>5.09 ± 0.24</td></tr></tbody></table>

Note: Values are expressed as Mean ± SEM; n=5. One-way ANOVA followed by Dunnett's t test. ***p < 0.001, **p < 0.01, *p < 0.05 is statistically significant with compared to control. MLGP = Methanolic leaves extract of G. pinnata; NHGP = N-hexane soluble fraction of G. pinnata leaves; EAGP = Ethyl acetate soluble fraction of G. pinnata leaves.

<figure><img src="image_10.png" /><figcaption>Fig. 6. Median lethal concentration (LC₅₀) of Vincristine sulfate, methanolic leaves extract, and its different fractions. MLGP = Methanolic leaves extract of G. pinnata; NHGP = N-hexane soluble fraction of G. pinnata leaves; EAGP = Ethyl acetate soluble fraction of G. pinnata leaves</figcaption></figure>

## 4. DISCUSSION

Medicinal plants are becoming increasingly crucial in novel drug discovery, as they contain unique chemicals with a range of activities that can treat various illnesses. To explore the novel therapeutic potentials of G. pinnata leaves and validate its traditional medicinal usage, several pharmacological activities of different fractions of this plant's leaves were scrutinised in this study. "The acetic acid-induced writhing test is a highly recommended model for evaluating the peripheral analgesic potentials of test compounds due to its sensitivity and capacity to

identify antinociceptive effects of natural products and test compounds at dose levels that are inert for other methods" (Yimer et al., 2020). When mice receive intraperitoneal acetic acid, the biosynthesis of prostaglandins via the arachidonic acid pathway generates pain, which is ultimately indicated as writhing reflexes (Islam et al., 2023). Our results showed that MLGP exerted a dose-dependent depletion in the percentage of inhibition of writhes in mice. Another two fractions, NHGP and EAGP, gave moderate effects. The data were highly significant (p<0.001) for both dosages of MLGP and EAGP 400.
The tail immersion testing approach is based on the findings that morphine-like substances selectively extend the time necessary for mice to react to the usual tail withdrawal reflex. An increase in the latency period or pain reaction time (PRT) signifies the degree of analgesia provided by the drug or extract (Ezeja et al., 2011). Investigations of this work unveiled that MLGP 400 exhibited the most significant (p<0.001) mean reaction time (7.25 ± 0.11 s) at 90 minutes. NHGP 400 and EAGP 400 also provided noteworthy responses simultaneously. We can assume from the findings of the acetic acid-induced writhing technique and tail immersion method that the extract exhibited its analgesic effect in both central and peripheral pathways. The phytochemical analysis confirmed the presence of flavonoids, tannins, phenols, and alkaloids, known for having analgesic potential. Flavonoids can block the synthesis of eicosanoids and arachidonic acid, while pyridine-based alkaloids provide antinociceptive effects.

"The castor oil-induced diarrheal model is a standard method for evaluating antidiarrheal medications. Castor oil contains ricinolic acid, an active metabolite that inhibits fluid and electrolyte absorption, leading to diarrhea" (Rahman et al., 2013). In this respect, our work revealed that several *G. pinnata* leaf fractions were significantly antidiarrheal in gastrointestinal tract alterations induced by castor oil. However, in the antidiarrheal models, the extract MLGP showed the most inhibitory activity. For the plant species to be employed in antidiarrheal formulations, the function, as mentioned earlier, must be explained together with the potential method by which the extract exerts effects.

The hypoglycemic assay demonstrated that, compared to the normal control group, the methanol extract of *G. pinnata* leaves (MLGP) at dosages of 200 and 400 mg/kg body weight considerably (p<0.001) lowered blood glucose levels at 60 minutes into the OGTT. When compared to the normal control group, the extract's significant (p<0.01) hypoglycemic effect persisted only at 120 minutes at the lower dose (200 mg/kg body weight). "The aqueous extract of *G. pinnata* bark was reported to have significant antidiabetic effects in a streptozotocin nicotinamide-induced type II diabetic rat model" (Shirwaikar et al., 2006). For the appropriate evaluation of the antidiabetic efficacy of *G. pinnata* leaves, extensive *in vivo* and *in vitro* investigations employing a variety of techniques are highly encouraged due to the paucity of

research on the hypoglycemic aspects of *G. pinnata* leaves. The presence of popular hypoglycemic phytocomponents such as flavonoids, tannins, and alkaloids has strengthened the findings of the *in vivo* assay.

The MLGP extract exhibited possible cytotoxic action in this investigation using the brine shrimp lethality assay (LC₅₀ = 52.58 µg/mL). Two more fractions (NHGP and EAGP) showed a moderate cytotoxic effect. These findings suggested that *G. pinnata* leaves had cytotoxic properties.

## 5. CONCLUSION

The current investigation highlighted the in vivo analgesic, antidiarrheal, hypoglycemic, and in vitro cytotoxic properties of the different fractions of *G. pinnata* leaves. There is currently little information on the bioactivity of this plant's leaves. Significant results from the antidiarrheal and analgesic tests support the utilisation of the herb for stomach problems and wound healing cases by the indigenous people. Furthermore, the fractions exhibited remarkable hypoglycemic and cytotoxic actions. Consequently, these findings foreshadow more research to gain a deeper understanding of the scientific process behind such acts.

## DATA AVAILABILITY STATEMENT

Data included in the article/supplementary material/referenced in the article.

## DISCLAIMER (ARTIFICIAL INTELLIGENCE)

Author(s) hereby declare that NO generative AI technologies such as Large Language Models (ChatGPT, COPILOT, etc) and text-to-image generators have been used during writing or editing of this manuscript.

## ACKNOWLEDGEMENTS

We are thankful to the Department of Pharmacy, Faculty of Biological Sciences, University of Chittagong, for the technical and laboratory support.

## COMPETING INTERESTS

Authors have declared that they have no known competing financial interests or non-financial interests or personal relationships that could have appeared to influence the work reported in this paper.
REFERENCES

Administration, U. S. F. and D. (2016). „FDA warns about serious heart problems with high doses of the antidiarrheal medicine loperamide (Imodium), including from abuse and misuse.

Ahsan, M. Q., Nasim, R., Nasim, M. T., & Islam, S. M. S. (2025). Anti-Inflammatory, Analgesic, and Anxiolytic Effects of Crude Extracts and Isolated Bioactive Fractional Compounds from Pouzolzia sanguinea. *Targets, 3*(2), 19.

Akhtar, M. S. (1992). Hypoglycaemic activities of some indigenous medicinal plants traditionally used as antidiabetic drugs. *Journal-Pakistan Medical Association, 42*, 271.

Alam, S., & Bhatnagar, S. (2006). Current status of anti-diarrheal and anti-secretory drugs in the management of acute childhood diarrhea. *The Indian Journal of Pediatrics, 73*(8), 693-696.

Almeida, C. E., Karnikowski, M. G. O., Foleto, R., & Baldisserotto, B. (1995). Analysis of antidiarrhoeic effect of plants used in popular medicine. *Revista de Saúde Pública, 29*, 428-433.

Arya, A., Abdullah, M. A., Haerian, B. S., & Mohd, M. A. (2012). Screening for hypoglycemic activity on the leaf extracts of nine medicinal plants: in-vivo evaluation. *Journal of Chemistry, 9*(3), 1196-1205.

Atta, A. H., & Mouneir, S. M. (2004). Antidiarrhoeal activity of some Egyptian medicinal plant extracts. *Journal of Ethnopharmacology, 92*(2-3), 303-309.

Awouters, F., Niemegeers, C. J. E., Lenaerts, F. M., & Janssen, P. A. J. (1978). Delay of castor oil diarrhoea in rats: a new way to evaluate inhibitors of prostaglandin biosynthesis. *Journal of Pharmacy and Pharmacology, 30*(1), 41-45.

Behzad, S., Pirani, A., & Mosaddegh, M. (2014). Cytotoxic activity of some medicinal plants from Hamedan district of Iran. *Iranian Journal of Pharmaceutical Research: IJPR, 13*(Suppl), 199.

Bhandari, R., Gyawali, S., Aryal, N., Gaire, D., Paudyal, K., Panta, A., Panth, P., Joshi, D. R., Rokaya, R. K., & Aryal, P. (2021). Evaluation of Phytochemical, Antioxidant, and Memory-Enhancing Activity of Garuga pinnata Roxb. Bark and Bryophyllum pinnatum (Lam) Oken. Leaves. *The

Scientific World Journal, 2021(1),
6649574.

Cragg, G. M., & Newman, D. J. (2000). Antineoplastic agents from natural sources: achievements and future directions. *Expert Opinion on Investigational Drugs, 9*(12), 2783-2797.

Ezeja, M. I., Omeh, Y. S., Ezeigbo, I. I., & Ekechukwu, A. (2011). Evaluation of the analgesic activity of the methanolic stem bark extract of Dialium guineense (Wild). *Annals of Medical and Health Sciences Research, 1*(1), 55-62.

Gawel, K., Jenda-Wojtanowska, M., Gibula-Bruzda, E., Kedzierska, E., Filarowska, J., Marszalek-Grabska, M., Wojtanowski, K. K., Komsta, L., Talarek, S., & Kotlinska, J. H. (2018). The influence of AMN082, metabotropic glutamate receptor 7 (mGlu7) allosteric agonist on the acute and chronic antinociceptive effects of morphine in the tail-immersion test in mice: Comparison with mGlu5 and mGlu2/3 ligands. *Physiology & Behavior, 185*, 112-120.

Hasan, Md. M., Azme, E., Alam, R., Mamun, Md. J. I., Chowdhury, Md. T., Rasel, Md. H., Momen, Md. S. H., Hoque, N., Ekram, Md. E. H., Eshaque, N. H., Ahmed, S., Tipu, Md. T. R., Juthi, S. S., Kabir, M. F., Ullah, A., Ali, Md. L., Hossen, S. M. M., & Chung, H.-J. (2025). Mushrooms as Potent Autophagy Modulators in Cancer Therapy: Current Evidence and Therapeutic Prospects. *Cancer Pathogenesis and Therapy*.
https://doi.org/https://doi.org/10.1016/j.cpt.2025.08.001

Hossain, S., Rabbi, S. A. H., Mamun, M. J. I., Masum, M. A. Al, Suma, K. J., Rasel, M. H., Hasan, M. A., Mohammad, M., & Hossain, D. (2025). Antioxidant, Anti-Inflammatory, and Neuropharmacological Potential of Syngonium podophyllum Flower Methanolic Extract: Insights From In Vivo, In Vitro, In Silico, and GC-MS/MS Analysis. *Chemistry & Biodiversity*, e202500425.

Hossen, S. M. M., Islam, M. J., Hossain, M. R., Barua, A., Uddin, M. G., & Emon, N. U. (2021). CNS anti-depressant, anxiolytic and analgesic effects of Ganoderma applanatum (mushroom) along with ligand-receptor binding screening provide new insights: Multi-disciplinary approaches. *Biochemistry and Biophysics Reports, 27*, 101062.
Islam, M. N., Devnath, H. S., Medha, M. M., Biswas, R. P., Biswas, N. N., Biswas, B., & Sadhu, S. K. (2023). In silico profiling of analgesic, antidiarrheal and antihyperglycemic properties of Tetrastigma bracteolatum (Wall.) leaves extract supported by in vivo studies. *Advances in Traditional Medicine, 23*(3), 789-801.

Jagannath, S., Vinayak, W., Amol, M., Sandhya, P., VM, C., Vaibhav, S., & Kulkarni, R. (2025). Molecular dynamics directed neuroprotective activity of alcoholic extract of Garuga pinnata Roxb. in experimental rats. *Journal of Ayurveda and Integrative Medicine, 16*(1), 101032.

Kirtikar, K. R., & Basu, B. D. (1991). Indian Medicinal Plants Periodical Expert Book Agency. *New Delhi, India, 3*, 2171-2172.

Kooti, W., Servatyari, K., Behzadifar, M., Asadi-Samani, M., Sadeghi, F., Nouri, B., & Zare Marzouni, H. (2017). Effective medicinal plant in cancer treatment, part 2: review study. *Journal of Evidence-Based Complementary & Alternative Medicine, 22*(4), 982-995.

Kupchan, S. M., & Tsou, G. (1973). Tumor Inhibitors. LXXXI. Structure and partial synthesis of fabacein. *The Journal of Organic Chemistry, 38*(5), 1055-1056.

Larner, J., & Haynes Jr, R. C. (1985). Insulin and oral hypoglycemic drugs; glucagon. In *The pharmacological basis of therapeutics* (pp. 1490-1503). MacMillan: New York.

Maikere-Faniyo, R., Van Puyvelde, L., Mutwewingabo, A., & Habiyaremye, F. X. (1989). Study of Rwandese medicinal plants used in the treatment of diarrhoea I. *Journal of Ethnopharmacology, 26*(2), 101-109.

Mekonnen, B., Asrie, A. B., & Wubneh, Z. B. (2018). Antidiarrheal activity of 80% methanolic leaf extract of Justicia schimperiana. *Evidence-Based Complementary and Alternative Medicine, 2018*(1), 3037120.

Mohammad, M., Chowdhury, Md. T., Eshaque, N. H., Mamun, Md. J. I., Rabbi, S. A. H., Hasan, Md. A., Chowdhury, M., Al-mamun, Md., Rashid, Md. J., & Hossen, S. M. M. (2025). Overdose Toxicological Effect of Methanol Extract of Popular Edible Colocasia esculenta Linn. Flowers: Biochemical, Hematological, and Behavioral Study on Swiss Albino Mice. *Food Science & Nutrition, 13*(7), e70674.

https://doi.org/10.1002/fsn3.70674

Mohammad, M., Islam, M. A., Mamun, M. J. I., Rasel, M. H., Masum, M. A. Al, Rabbi, S. A. H., Khalil, I., Ali, M. L., & Hossen, S. M. M. (2025). Methanolic Extract of Edible Lasia spinosa Rhizome: A Potential Natural Source of Analgesic, Diuretic, and Thrombolytic Agents. *Journal of Herbs, Spices & Medicinal Plants, 31*(3), 381-406.

Mohammad, M., Mamun, M. J. I., Khatun, M., Rasel, M. H., Masum, M., Suma, K. J., Haque, M. R., Rabbi, S. A. H., Hossain, M. H., & Hasnat, H. (2025). A Multifaceted Exploration of Shirakiopsis indica (Willd) Fruit: Insights into the Neuropharmacological, Antipyretic, Thrombolytic, and Anthelmintic Attributes of a Mangrove Species. *Drugs and Drug Candidates, 4*(3), 31.

Mohammad, M., Rasel, M. H., Richi, F. T., Mamun, M. J. I., Ekram, M. E. H., Rabbi, S. A. H., Hossain, S., Hasan, M. A., Sarker, M. F., & Alam, S. (2025). Neuropharmacological, cytotoxic, and anthelmintic potentials of Lasia spinosa (L.) Thwaites rhizome: in vivo, in vitro, and computational approach. *Pharmacological Research-Natural Products, 100*254.

Nadkarni, A. K. (2007). *Dr. KM Nadkarni's Indian materia medica: with Ayurvedic, Unani-tibbi, Siddha, allopathic, homeopathic, naturopathic & home remedies, appendices & indexes* (Vol. 1). Popular Prakashan.

Nahar, A., Jahin Khandakar, M., Jahirul Islam Mamun, M., Hossain Rasel, M., Bin Ihsan, A., Raj, A., Ahmed, S., Kamrul Hossain, M., Riasat Hasan, M., & Saito, T. (2025). Investigation of Analgesic, Anti-Inflammatory, and Thrombolytic Effects of Methanolic Extract and Its Fractions of Dischidia bengalensis: In Vitro and In Vivo Studies with In Silico Interventions. *Molecules, 30*(18), 3724. https://doi.org/10.3390/molecules3018372

Oecd, O. (2001). *Guidelines for the Testing of Chemicals. Acute Oral Toxicity—Acute Toxic Class Method: Test No-423 Organization for Economic Co-Operation and Development.*

Quader, J. M. B., Nisat, U. T., Nath, A. K., Barua, C., Bristy, K. F., Sultana, M., Rahman, M., & Uddin, M. (2024). Comprehensive Pharmacological Assessment and Phytochemical Analysis of Tetrastigma
angustifolia (Roxb.) Deb.: Unveiling
Analgesic, Anxiolytic and Hypoglycemic
Potential. *Asian Journal of Research in
Botany*, 7(2), 235-246.

Rahman, M. A., Sultana, R., Akter, R., & Islam, M. S. (2013). Antidiarrheal and antidiabetic effect of ethanol extract of whole Ageratum conyzoides L. in albino rat model. *African Journal of Pharmacy and Pharmacology*, 7(23), 1537-1545.

Shirwaikar, A., Rajendran, K., & Barik, R. (2006). Effect of aqueous bark extract of Garuga pinnata Roxb. in streptozotocin-nicotinamide induced type-II diabetes mellitus. *Journal of Ethnopharmacology*, *107*(2), 285-290.

Shirwaikar, A., Rajendran, K., & Barik, R. (2007). Effect of Garuga pinnata. Roxb Aqueous Bark Extract on Tissue Antioxidant Activity in Streptozotocin-Nicotinamide-Induced Type II Diabetic Rats. *Pharmaceutical Biology*, *45*(3), 205-209.

Tareq, A. M., Farhad, S., Uddin, A. B. M. N., Hoque, M., Nasrin, M. S., Uddin, M. M. R., Hasan, M., Sultana, A., Munira, M. S., & Lyzu, C. (2020). Chemical profiles, pharmacological properties, and in silico studies provide new insights on Cycas pectinata. *Heliyon*, 6(6).

Thupurani, M. K., Reddy, P. N., Singara Charya, M. A., & Geethanjali, M. (2013). In vitro and in vivo determination of anti-inflammatory activities of Garuga pinnata Roxb. *Int J Pharm Sci Rev Res*, 22(2), 310-314.

Thupurani, M. K., Reddy, P. N., Thirupathaiah, A., Charya, M. A. S., & Shiva, D. (2012). *In vitro* determination of anti-oxidant activities of *Garuga pinnata* Roxb.

Yimer, T., Birru, E. M., Adugna, M., Geta, M., & Emiru, Y. K. (2020). Evaluation of analgesic and anti-inflammatory activities of 80% methanol root extract of Echinops kebericho M. (Asteraceae). *Journal of Inflammation Research*, 647-658.
# SUPPLEMENTARY MATERIALS

## 1. ANALGESIC ACTIVITY

### 1.1 Acetic Acid-Induced Writhing

<table><thead><tr><th rowspan="2">Animal Group</th><th colspan="5">Writhing Count</th><th rowspan="2">Mean ± SEM</th><th rowspan="2">% Inhibition</th></tr><tr><th>M-1</th><th>M-2</th><th>M-3</th><th>M-4</th><th>M-5</th></tr></thead><tbody><tr><td>Control</td><td>41</td><td>36</td><td>44</td><td>38</td><td>42</td><td>40.2 ± 1.43</td><td>-</td></tr><tr><td>Standard</td><td>18</td><td>16</td><td>16</td><td>15</td><td>15</td><td>16 ± 0.55</td><td>59.70</td></tr><tr><td>MLGP 200</td><td>31</td><td>25</td><td>28</td><td>33</td><td>21</td><td>27.6 ± 2.14</td><td>31.34</td></tr><tr><td>MLGP 400</td><td>21</td><td>18</td><td>28</td><td>25</td><td>22</td><td>22.8 ± 1.71</td><td>43.28</td></tr><tr><td>NHGP 200</td><td>41</td><td>34</td><td>31</td><td>38</td><td>36</td><td>36 ± 1.70</td><td>10.45</td></tr><tr><td>NHGP 400</td><td>35</td><td>29</td><td>28</td><td>31</td><td>38</td><td>32.2 ± 1.88</td><td>19.90</td></tr><tr><td>EAGP 200</td><td>35</td><td>29</td><td>39</td><td>25</td><td>31</td><td>31.8 ± 2.42</td><td>20.90</td></tr><tr><td>EAGP 400</td><td>31</td><td>28</td><td>25</td><td>29</td><td>32</td><td>29 ± 1.22</td><td>27.86</td></tr></tbody></table>

## 2. CYTOTOXIC ACTIVITY

### 2.1 Brine Shrimp Lethality Assay

#### Vincristine Sulphate (Standard)

<table><thead><tr><th>Concentration (µg/mL)</th><th>Number of Nauplii Dead</th><th>% of mortality</th><th>LC<sub>50</sub> (µg/mL)</th></tr></thead><tbody><tr><td>20</td><td>10</td><td>100</td><td>1.715</td></tr><tr><td>10</td><td>9</td><td>90</td><td></td></tr><tr><td>5</td><td>7</td><td>70</td><td></td></tr><tr><td>2.5</td><td>6</td><td>60</td><td></td></tr><tr><td>1.25</td><td>6</td><td>50</td><td></td></tr><tr><td>0.625</td><td>3</td><td>40</td><td></td></tr><tr><td>0.3125</td><td>2</td><td>30</td><td></td></tr></tbody></table>

#### MLGP

<table><thead><tr><th>Concentration (µg/mL)</th><th>Number of Nauplii Dead</th><th>% of mortality</th><th>LC<sub>50</sub> (µg/mL)</th></tr></thead><tbody><tr><td>400</td><td>10</td><td>7</td><td>52.58</td></tr><tr><td>200</td><td>8</td><td>7</td><td></td></tr><tr><td>100</td><td>9</td><td>6</td><td></td></tr><tr><td>50</td><td>7</td><td>4</td><td></td></tr><tr><td>25</td><td>5</td><td>5</td><td></td></tr><tr><td>12.5</td><td>3</td><td>5</td><td></td></tr><tr><td>6.25</td><td>1</td><td>2</td><td></td></tr></tbody></table>

#### NHGP

<table><thead><tr><th>Concentration (µg/mL)</th><th>Number of Nauplii Dead</th><th>% of mortality</th><th>LC<sub>50</sub> (µg/mL)</th></tr></thead><tbody><tr><td>400</td><td>8</td><td>9</td><td>113.39</td></tr><tr><td>200</td><td>10</td><td>8</td><td></td></tr><tr><td>100</td><td>7</td><td>6</td><td></td></tr><tr><td>50</td><td>6</td><td>3</td><td></td></tr><tr><td>25</td><td>5</td><td>2</td><td></td></tr><tr><td>12.5</td><td>1</td><td>2</td><td></td></tr><tr><td>6.25</td><td>2</td><td>1</td><td></td></tr></tbody></table>
EAGP

<table><thead><tr><td>Concentration (µg/mL)</td><td>Number of Nauplii Dead</td><td>% of mortality</td><td>LC<sub>50</sub> (µg/mL)</td></tr></thead><tbody><tr><td>400</td><td>9</td><td>9</td><td>129.28</td></tr><tr><td>200</td><td>5</td><td>8</td><td>70</td></tr><tr><td>100</td><td>6</td><td>8</td><td>60</td></tr><tr><td>50</td><td>4</td><td>6</td><td>50</td></tr><tr><td>25</td><td>4</td><td>3</td><td>30</td></tr><tr><td>12.5</td><td>4</td><td>1</td><td>20</td></tr><tr><td>6.25</td><td>0</td><td>3</td><td>10</td></tr></tbody></table>

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