Assessment of Biological Activities, Acute and Sub-chronic Toxicity of Liang (Gnetum gnemon var. tenerum) Leaves Powder, a Natural Product

Abstract

Gnetum gnemon var. tenerum (Gnetaceae) is a shrub plant native to South-East Asia. In Thailand, Liang leaves are commonly consumed in South of Thailand as vegetable. According to literature, they have an antihyperglycemic capacity because of their rich chlorophyll, fiber, and protein. However, there is need to assess the safety since natural food products are not completely devoid of toxicity. This study aimed to assess the biological activities as well as the acute and sub-chronic oral toxicity of Liang leaves powder (LLP). The evaluation of LLP for acute oral toxicity was performed at dose level 2000 mg/kg body weight in Wistar rats while the sub-chronic oral toxicity of LLP was performed at the effective dose (1.47 g/kg) for antihyperglycemic property according to Organisation for Economic Co-operation and Development (OECD)-425. The results showed that LLP demonstrated anti-inflammatory activities. It also showed no clinical signs of toxic effects and mortality in rats throughout 90 d. Thus, LLP could be classified in GHS category 5 which are of relatively low acute toxicity and then the lethal dose, 50% (LD₅₀) cut off at 5000 mg/kg body weight to infinity (∞). Administration of LLP to the experimental rats significantly increased (p < 0.05) the concentration of triglyceride and increased concentration of creatinine as a result of kidney malfunction was also noticed in the experimental rats. Hematological alteration was not noticed in the treated female rats, but red blood cell, hemoglobin and hematocrit concentrations significantly increased in the treated male rats. The study concludes that sub-chronic administration of 1.47 g/kg LLP is relatively safe.

INTRODUCTION

The awareness of natural food products and healthy food is increasing among the global populace.¹⁾ Also, over the past decades, herbal medicine and/or natural food products are being used to manage various diseases including gastrointestinal, respiratory, obesity, hypertension, diabetes, etc. based on the belief that plants are accessible, cheaper, and natural remedy without any side effects. Gnetum gnemon is a shrub plant that is native to South-East Asia belonging to the Gnetaceae family. In Africa, there are over 30 species of Gnetum plant in existence.²⁾ It is commonly called Gnetum or Two Leaf Tree in English³⁾ and with varying local names in different countries. Ethnobotanical study revealed that Gnetum is one of the oldest plant species to ever exist on Earth planet dating back to more than 200 million years. All parts of the plant are useful and beneficial while the fruits, seeds and leaves are the most edible.⁴⁾ G. gnemon is considered as a candidate plant for food, nutraceuticals, and medicinal products because of its high nutritional value.⁵⁾ In New Guinea, the leaves are cooked with sauce of other plants and eaten with meat.⁶⁾ Also in Indonesia, India and East Java, the leaves, fruits, and seeds are processed into different forms, cooked with other recipes, and consumed as human food.²⁾ Gnetum gnemon var. tenerum is the most popular variety of gnemon species in Thailand⁷⁾ where is locally called Liang. Liang leaves are consumed as human food vegetables in Southern Thailand⁸⁾ and more recently it has been sold as commercial menu in a major supermarket of the country.⁵⁾ Generally, G. gnemon is planted as co-plant or intercropping as well for food and energy purposes. More recently, the Thai government provided support grants to conduct research on the plants owing to its benefits. Many chemical constituents including saponins, phenolic compounds, alkaloids, vitamins A, C, fatty acids, amino acids, beta-carotene among others have been identified in G. gnemon leaves.^5,9,10)

These chemical constituents have been reported to play significant role in the biological activities such as antioxidant,¹¹⁾ anti-asthmatic and anti-inflammatory activity,¹²⁾ acetylcholine inhibitory activity, antimicrobial activity, anti-plasmodial,¹³⁾ anti-acne activity,¹⁴⁾ anti-cancer and anti-tumor activities,¹⁵⁾ anti-hypertensive activity^16,17) and anti-diabetic activity¹⁸⁾ demonstrated by the plant. Studies on nutritional value also revealed that G. gnemon leaves are rich sources of protein and dietary fiber (27 and 36.3% in g/100 g, respectively,⁵⁾ as well as chlorophyllin; a chlorophyll derivative known to inhibit gluconeogenesis and enhance cellular uptake of glucose³⁾ which is one major reason G. gnemon var. tenerum is highly consumed as a natural food in South of Thailand for diabetic therapy.¹⁸⁾ These compounds play key roles in regulating blood sugar. Despite the health benefits of Liang leaves, there is need to investigate the safety since natural food products may not be completely devoid of side effects and toxicity. Additionally, toxicity information of Liang leaves was scarce and insufficient to confirm Liang leaves powder (LLP) is safe for human administration in short and long term consumption. Therefore, this study was conducted to investigate the acute and sub-chronic toxicity of LLP following Organisation for Economic Co-operation and Development (OECD)-425 and OECD-408 on abnormalities such as mortality, clinical signs of toxic, body and organ weight change, as well as biochemical, hematological, and histological in rats to promote awareness about the safety of using this plant.

MATERIALS AND METHODS

Plant Collection and Powder Preparation

The fresh Liang (Gnetum gnemon var. tenerum) leaves were harvested from the farm of Mr. Chokdee Sangkaew located at House No. 6, Village No. 6, Thapo Sub-District, Sadao District, Songkhla Province, Thailand in February 2022. After being washed with a 100 ppm chlorinated solution to remove contaminants, the leaves were dried using a vacuum-microwave set at 3600 W, with 15 min of drying time per 0.5 kg. The dried leaves were then pulverized into a fine powder, sieved with a 280 mesh sieve, and stored in an aluminum foil zipper bag at −20 °C in the refrigerator until needed. The total yield from harvest to powder was 12.46%.

Preparation of LLP Stock Solution

For acute toxicity assessment, the LLP was administered at the dose of 2000 mg/kg body weight. In the sub-chronic toxicity assessment, the dose of LLP at 1.47 g/kg body weight according to the effective dose for anti-hyperglycemic in previous study. The LLP was freshly mixed with distilled water and glycerol (1 : 6 v/v) to suspend it as a vehicle before administration. The constant volume did not exceed 1 mL per 100 g of animal body weight.

Experimental Animals and Handling

In acute toxicity assessment, a total of ten (10) female Wistar rats (Rattus novergicus) which weighed 180–200 g and aged 8 weeks were recruited for the study and divided into 2 groups including control group (N = 5) and treated group (N = 5). In sub-chronic toxicity assessment, twenty male and twenty female of Wistar rats which 150–250 g and 5 weeks were used. They were purchased from Nomura Siam International, Thailand and raised at the Laboratory Animal Center, Prince of Songkla University, Thailand where they were acclimatized under normal condition of temperature 22 ± 3 °C, relative humidity 55 ±  10%, light intensity 130–325 lx, lighting period 12/12 h (bright/dark) and filtered water through reverse osmosis system before the experiment. Rats had free access to enough water and food (rat pellets) obtained commercially.

Biological Activities Test of LLP

Anti-hyperglycemic Activity

Anti-hyperglycemic was determined through decreasing of non-fasting blood glucose (NFBG), glycated haemoglobin (HbA1C) level, glucose transporter 2 (GLUT2) and GLUT4 expression between normal rats, diabetic rats, and treated rats with 1.47 g/kg body weight of LLP on Wistar rat. NFBG levels were assessed on the 1st, 3rd, 7th, 14th, 21st, and 28th days to determine anti-hyperglycemic activity. At the end of the study, blood was collected by cardiac puncture to determine the HbA1c level using MISPA I3 Automated Cartridge Based Specific Protein Analyzer (Agappe Hills, Kerala, India). The liver specimen was used to determine GLUT2 while adipose tissue was used to determine of GLUT4 expression using quantitative real-time PCR (qPCR) by CFX96 Touch Real-Time PCR Detector (Bio-Rad Laboratories, CA, U.S.A.) with 5× HOT FIREPol^® EvaGreen^® qPCR Mix Plus (ROX) (Solis BioDyne, Tartu, Estonia).

Phenolic Content and Antioxidant Study

LLP was boiled in water at 80 °C for 30 min at a ratio of 1 : 20 and the supernatant was collected using Buchner funnel and then centrifuged at 4 °C for 20 min at 7100×g.¹⁹⁾ The total phenolic content and antioxidant activity of LLP was determined using Ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picryl hydrazyl (DPPH) and 2,2-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical scavenging assay.

1) Total phenolic content (TPC) determination

TPC was determined by the method described by Singleton and Rossi.²⁰⁾ Briefly, 20 µL of the LLP extract was added to a 96-well plate, followed by the addition of 100 µL of 10% Folin reagent (v/v). The mixture was incubated in the dark at 30 °C for 6 min. Afterward, 7.5% Na₂CO₃ (anhydrous) (w/v) was added, and the mixture was further incubated for 30 min. The absorbance of the resulting solution was added at 765 nm using a microplate reader (Varioskan LUX, Thermo Scientific, Singapore). Gallic acid and Trolox were used as standard compounds. Standard curves were constructed using different concentration ranges: 0–200 µg/mL for gallic acid and 0–500 µg/mL for Trolox.

2) FRAP assay

The FRAP assay was conducted in accordance with the method outlined by Benzie and Strain.²¹⁾ A freshly prepared FRAP solution consisting of 300 mM acetate buffer (pH 3.6), 10 mM TPTZ (2, 4, 6-tripyridyl-s-triazine) in 40 mM HCl, and 20 mM FeCl₃·6H₂O (in a ratio of 10 : 1 : 1) was heated at 37 °C for 30 min. Subsequently, 15 µL of the LLP extract was combined with 285 µL of the FRAP solution and incubated at 37 °C for 30 min. The absorbance of the resulting mixture was measured at 593 nm.

3) DPPH radical scavenging activity

The DPPH radical scavenging activity was evaluated following the method outlined by Brand-Williams et al.²²⁾ Gallic acid and Trolox were used as standard compounds. Standard curves were constructed using different concentration ranges: 0–5 µg/mL for gallic acid and 0–10 µg/mL for Trolox. Initially, 100 µL of the LLP extract was mixed with 100 µL of 0.2 mM DPPH in 95% ethanol. The resulting mixture was then incubated in the dark for 30 min at 30 °C. Finally, the absorbance was measured at 517 nm.

4) ABTS radical scavenging activity

The ABTS assay was conducted following the method described by Arnao et al.²³⁾ Gallic acid and Trolox were used as standard compounds. Standard curves were constructed using different concentration ranges: 0–20 µg/mL for gallic acid and 0–0.75 µg/mL for Trolox. To generate the ABTS radical, a 7.4 mM ABTS solution was incubated in the dark at 30 °C for 12 h. Subsequently, 20 µL of the LLP extract was mixed with 280 µL of the radical solution and kept in the dark for 2 h at 30 °C. Finally, the absorbance of the mixture was measured at 734 nm.

Cell Toxicity of Macrophages Cell Cultures by MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide)

Murine macrophage or RAW264.7 was cultured in RPMI-1640 (Roswell Park Memorial Institute) consist of 10% fetal bovine serum (FBS), 100 U/mL of penicillin and 100 µg/mL of streptomycin with 5% CO₂ (CO₂ incubator) at 37 °C. After cell growth reached 80% of tissue culture ware 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) solution was used to wash cell from tissue culture ware and count to 5 × 105 cell/mL. One hundred microliter of cell solution was pipetted into each well of 96 well plate and incubated for 2 h. RPMI-1640 was replaced with 100 µL of sample solution at the concentration 6.25, 3.125, 1.563, and 0.781 mg/mL was added to each well (RPMI-1640 100 µL using as standard) and incubated for 24 h. Thereafter, 10 µL MTT solution at concentration 5 mg/mL was added to 96 well plate and incubated 37 °C for 2 h. All solution was replaced with 200 µL dimethyl sulfoxide (DMSO) to dissolve formazane crystal and read at 570 nm. Cell viability was calculated as follows and concentration with cell viability higher than 80% was selected for determined anti-inflammation properties²⁴⁾ (Equation 1).

  

(1)

• O.D._Sample means absorbance of sample
• O.D._Control means absorbance of standard (RPMI)

Determination of Anti-inflammatory Properties by Nitric Oxide Inhibition Assay

Murine macrophage (RAW264.7) was cultured to 80% of tissue culture ware. After cell growth reach 80% of tissue culture ware 0.25% trypsin–EDTA solution was used to wash cell from tissue culture ware and count to 5 × 105 cell/mL. One hundred microliters of cell solution was pipetted into each well of 96 well plate and incubated for 2 h. Then RPMI-1640 was replaced with 200 µg/mL lipopolysaccharides (LPS) solution with RPMI-1640 per well (RPMI-1640 100 µL using as standard). After that, 100 µL of sample solution of LLP at the concentration 6.25, 3.125, 1.563, and 0.781 mg/mL l per well was added to 96-well plate except control and blank using 100 µL of RPMI and incubated at 37 °C for 24 h. After incubated, 100 µL of each well was transfer to new 96-wells plates for anti-inflammation determination using 100 µL Griess reagent per well. Spectrophotometry at 570 nm was used to read 96 well plate.²⁴⁾ Inhibition of nitric oxide (NO) production (%) was calculated as followed (Equation 2).

  

(2)

• ODC means absorbance of control (RPMI + LPS)
• O.D._Bc means absorbance of blank (RPMI)
• O.D._S means absorbance of sample solution (Sample + LPS)
• O.D._Bs means absorbance sample blank (Sample + RPMI)

Grouping and Administration Dosage of LLP

The Acute Toxicity Assessment

The acute toxicity assessment of the LLP was administered at the dosage of 2000 mg/kg body weight. The animals were divided into two groups (n = 5 each). G1, the control group, which received only distilled water; and G2, the treated group, received a single dose of 2000 mg/kg of LLP. After 12 h of fasting, with only access to the water, the rats were weighed and administered LLP orally via orogastric canulla. In the first day of experiment, one rat of treated group (G2) at the dose of 2000 mg/kg LLP was orally administration, if no shown evidence toxic symptoms within 24 h, new 4 rats of group 2 were repeated at the dose of 2000 mg/kg of LLP. Toxic symptoms were observed for the first 30 min, then for 4 h, and every 24 h for 14 d. The rats were fed food pellets and water throughout the experiment, and their respiration, salivation, feces consistency, mucous membranes, skin, coat changes, sedation, seizures, lethargy, and coma were all monitored.

The Sub-chronic Toxicity Assessment

The test dose of 1.47 g/kg body weight for LLP which is the effective dose for the antihyperglycemic study was used to assess the sub-chronic toxicity of LLP in rats. Twenty male and twenty female Wistar rats were randomized into 4 groups of 10 rats and treated as follows:

• Group A: Control male rat which received the distilled water.
• Group B: Treated male rat which received the LLP 1.47 g/kg b.w. of LLP
• Group C: Control female rat which received the distilled water.
• Group D: Treated female rat which received the LLP 1.47 g/kg b.w. of LLP

Rats of the same sex were housed together. They were fasted for 12 h and their weight was taken.

This was followed by daily oral gavage of 1.47g/kg b.w. of LLP extract to rats every day up to 90 d. On the last day of experiment, rats were fasted overnight and weighed again before euthanized.

Necropsy Examination, Clinical Biochemistry, and Hematological Analysis

On the final day of the experiment, the rats subjected to acute and sub-chronic toxicity test respectively were euthanized using 120 mg/kg of sodium thiopental after fasting. Blood samples were obtained via cardiac puncture for further analysis. Biochemical parameters indicative of liver and renal functions such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatinine were determined from serum of rats using standard Kit procedure with the Mindray BS-200 clinical analyzer. Hematological parameters including red blood cell (RBC), white blood cell (WBC), platelet counts, hemoglobin (Hb) level, hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were assessed from the plasma of rats using the Nihon Kohden Celltac Es MEK-7300 automated hematology analyzer.

Histopathological Study

In acute and sub-chronic toxicity, histopathological examination was performed on the liver and kidney. The histopathological analysis involved suspending the organs in 10% formalin for 3 d, followed by dehydration and embedding them in paraffin wax (5 µm sections). Subsequently, the slides were stained with hematoxylin and eosin, and structural changes were examined under a light microscope.

Organ Weighting

After euthanasia, heart, kidney, spleen, and liver were collected and rinsed with normal saline and dried with tissue paper. Organ weight was measured with precision balance for calculation of organ index as shown in Equation 3.

  

(3)

Ethical Approval

Ethical authorization on the use of experiment animals for this study was issued by Institutional Animal Care and Use Committee, Prince of Songkla University Thailand with Ref. No. 55/2021.

Statistical Evaluation

Data were presented as mean ± standard error of the mean (S.E.M.). Statistical evaluation between the control and treated group was performed using ANOVA followed by t-test with SPSS software (Version 28.0). Values were considered statistically significant at p < 0.05.

RESULTS AND DISCUSSION

Biological Activities of Liang Leaf Powder

Total Phenolic Content of LLP

Table 1 shows the TPC of LLP. The TPC was found to be 3548.78 ± 346.25 µg/g Gallic acid equivalent dry weight and 14985.93 ± 1488.46 µg/g Trolox equivalents, respectively. This value of TPC reported for LLP in this study is quite high and therefore corroborates literature report that vegetables are good sources of phenolic and compounds.

Table 1. Total Phenolic Content and Antioxidant Activities of Liang Leaves Powder

Parameters	Standard
	Gallic acid (µg/g) DW	Trolox (µg/g) DW
Total phenolic content (TPC)	3548.78 ± 346.25	14985.93 ± 1488.46
Ferric reducing antioxidant power (FRAP)	192.79 ± 18.90	1,248.14 ± 115.12
DPPH radical scavenging activity	152.90 ± 7.83	246.02 ± 12.29
ABTS radical scavenging activity	767.21 ± 40.66	3247.29 ± 169.65

Antioxidant Activity

The antioxidant activity of LLP is depicted in Table 1. The LLP demonstrated higher radical scavenging activity for ABTS followed by FRAP and then least activity for DPPH with respect to the standard antioxidant’s compounds (Gallic and Trolox) employed in this study. The antioxidant activity demonstrated by LLP in scavenging ABTS, DPPH as well as ferric ion reflects the phenolic compounds likely to be present in the plant which could be responsible for this antioxidant property displayed by LLP. This implies that they are able to donate electrons to radical compounds such as ABTS and DPPH and convert them from a radical form to a non-radical form.²⁵⁾ Phenolic compounds and flavonoids are believed to have therapeutic benefit against different diseases caused by oxidative stress, one of which is diabetes.²⁶⁾

Anti-hyperglycemic

Figures 1A and B demonstrate the anti-hyperglycemic activity of LLP in Wistar rats. After 28 d of treatment, rats that received 1.47 g/kg of LLP (DM/LLP 1.47 g/kg) exhibited a reduction of approximately 36.12% in NFBG compared to diabetic rats (DM). Moreover, the administration of 1.47 g/kg of LLP resulted in a decrease in HbA1C levels by approximately 25.51% compared to diabetic rats. This finding is consistent with the study by Abani et al.,²⁷⁾ which reported that chlorophyllin (CHL) can lower blood glucose levels, and its mechanism of action is similar to that of metformin, the standard medicine for diabetic patients. Anisong et al.⁵⁾ indicated that LLP is a rich source of chlorophyll. Despite this knowledge, the exact mechanism underlying the use of a high dose of LLP (1.47 g) to decrease NFBG and HbA1c in rats remains unclear. However, it is hypothesized that this effect may be attributed to the uptake of chlorophyll present in LLP by hepatocytes. The chlorophyll in LLP could accumulate in the mitochondria and inhibit mitochondrial complex 1, resulting in a decrease in ATP production. When ATP levels decrease, AMP levels increase, which promotes glucose uptake and inhibits gluconeogenesis.²⁸⁾

Fig. 1. Biological Activities of Liang Leaves Powder

(A) Effects of Liang leaves powder on non-fasting blood glucose. (B) The change of HbA1c level by Liang leaves powder, (C) Effects of Liang leaves powder on GLUT2 gene expression, (D) Effects of Liang leaves powder on GLUT4 gene expression. DM was diabetic rat group; and DM/CLL 1.47 g was diabetic rat group treated with 1.47 g/kg of crude Liang leaves powder (mean ± S.E.M., n = 6), lowercase letters indicate mean significantly different with in a day (p < 0.05).

Figures 1C and D illustrate the effect of LLP on the expression of GLUT2 and GLUT4. The results showed that the administration of LLP led to a reduction in GLUT2 expression and an increase in GLUT4 expression. GLUT2 is a transporter present in the membranes of hepatocytes and pancreatic beta cells which regulates insulin secretion and glucose balance.²⁹⁾ In diabetic conditions, particularly type 2 diabetes, insulin secretion and GLUT2 expression are typically elevated due to the storage of excess glucose in hepatocytes from bloodstream.³⁰⁾ Following the administration of LLP to diabetic rats for 28 d in this study, we noticed GLUT2 expression decreased by approximately 57.35% thus suggesting the gluconeogenesis inhibition potential of LLP in hepatocytes as well its potential for insulin regulation in beta cells. Solaimani³¹⁾ discussed how glucose uptake can be measured through GLUT4 expression in adipose and muscle tissues. From our observation, the administration of LLP resulted in approximately a four-fold increase in GLUT4 expression when compared to the diabetic group. This implies that LLP could facilitate glucose uptake in adipose tissue and muscle cells, leading to a decrease in blood glucose levels. In general, LLP appears to contribute to improved glucose uptake in adipose tissue and muscle cells which ultimately resulted in the reduction of NFBG as evident in this study.

Inflammatory Response

Nitric Oxide Inhibition and Cell Viability

The effect of LLP on nitric oxide inhibition and cell viability in vitro is shown in Fig. 2. The results showed that LLP exhibited a dose-dependent inhibitory effect on nitric oxide production in vitro. The highest nitric oxide inhibition (90%) for LLP was recorded at the highest concentration of 6.25 while the lowest inhibition of nitric oxide production (25%) was recorded at the lowest concentration of 0.781 for LLP. In a similar manner, the ability of LLP to enhance cell viability in vitro was also concentration-dependent however contrary to what was observed on the inhibition of nitric oxide, the highest cell viability (95%) was recorded at the least concentration of 0.781 while the lowest cell viability (75%) was recorded at highest concentration of 6.25. Cytotoxicity assay which measures cell viability is a method used to indicate the potentiality of an agent in causing raw cell line destruction. High percentage of cell viability corresponds to a lower cytotoxicity effect of an agent and vice-visa. It is evident from this study that the LLP does not pose any significant toxic effect on cell since over 70% cell viability was achieved even at the minimum concentration. Higher NO production as a reactive oxygen species is associated with inflammation.³²⁾ Therefore, reduction or inhibition of NO production is an essential parameter for inflammation determination. It is also evident from our study that LLP can mediate inflammatory response since up to 90% inhibition of NO production was achieved at the highest concentration. This inflammatory response demonstrated by LLP perhaps could be due to its phenolic composition contained in this leafy vegetable since vegetables are rich sources of phenolic compounds and have been reported to demonstrate anti-inflammatory activity.³³⁾

Fig. 2. Anti-inflammation Activity of Liang Leaves Powder

(A) Effects of Liang leaves powder on nitric oxide inhibition. (B) Effects of Liang leaves powder on cell viability.

Acute Toxicity Test of LLP in Wistar Rats

Following the oral administration of 2000 mg/kg b.w. LLP to evaluate the acute toxicity for 14 d using OECD-425 guideline, it was observed that treatment of rats with Liang leaves did not express any symptoms of toxicity or mortality after oral administration at a dose of 2000 mg/kg. Normal behavior, respiration, salivation, feces consistency, mucous membrane, skin and coat changes, sedation, seizures, lethargy, and coma of both groups (G1 and G2) were similar as they did not show any abnormal sign.

The Sub-chronic Toxicity of LLP in Wistar Rats

Following the oral administration of 1.47 g/kg LLP to evaluate the sub-chronic toxicity for 90 d using OECD-408 guideline, it was observed that treatment of rats with Liang leaves did not express any symptoms of toxicity or mortality after oral administration of LLP in both sexes of rats. The rats which received the LLP for 90 d were 100% survivors with normal phenomenon.

In addition, all rats in both groups showed normal behavior, respiration, salivation, feces consistency, mucous membrane, skin and coat changes during the experiment. No sedation, seizures, lethargy, and coma were noticed among rats. Toxicological study in experimental animals is the most reliable method of detecting important toxic properties of chemical substances and for estimating risks to human.³⁴⁾ Test substances that are toxic to animals manifest clinical signs and abnormal behavioral changes which may include convulsions, tremors, liquefaction, dexterity, pallor and moistness of the nasal mucosa and eyes as well as death.³⁵⁾ In general, the animals understudied for acute and sub-chronic toxicity in this study showed no symptoms of toxicity and mortality upon receiving 2000 mg/kg and 1.47 g/kg respective doses of LLP. This suggests that Liang leaf is safe at the investigated doses. This finding corroborates the earlier report of Tatefuji et al.³⁶⁾ on Gnetum gnemon seed powder where the authors established that the seed powder is safe as food consumption in Indonesia at the investigated acute dose.

Body Weight, Growth Rate, and Organ Weight

Figures 3A and B show the body weight and growth rate of the control and treated animals for acute toxicity study. Treatment with 2000 mg/kg of Liang (Gnetum gnemon var. tenerum) leaves powder did not cause any significant change (p > 0.05) in the body weight of the rats in comparison to the control group. Although, there was a progressive increase in body weight observed on the 7th day in both the control and treated groups, with a growth rate of 6.94 and 5.77%, respectively. On the last day of the experiment, a further increase in body weight was recorded in both groups, at growth rates of 10.58 and 15.35%, respectively; however, these were not significantly different (p > 0.05). The body weight and organ weight of the rats treated with 2000 mg/kg LLP were not significantly different from the control rats, despite the increase in the growth rate of the individual group, indicating that Liang leaves did not induce any growth abnormalities. The increase in growth rate observed on 7th and 14th day in the acute toxicity test group that received LLP implies that the LLP contains compounds particularly protein that promotes growth. Recent study by Anisong et al.⁵⁾ reported that LLP contained 25.5 g protein in 100 g of the dried leaf as well as essential and branch chain amino acids. Figures 3C–F showed the effect of LLP administration on the organ-to-body weight index of the experimental rats. There was no significant difference (p > 0.05) in the ratio of organ (liver, kidney, heart, and spleen) to body weight in rats treated with LLP at 2000 mg/kg orally when compared to the control rats.

Fig. 3. Effect of the Liang Leaves Powder on Body Weight and Organ to Body Weight in Rats

(A) Body weight was monitored on 1st, 7th, and 14th days, (B) Growth rate observed on 7th and 14th days, (C) Liver index, (D) Kidney Index, (E) Spleen index, (F) Heart index. Values with same alphabet are not statistically different (p > 0.05) (n = 5).

In the sub-chronic toxicity test of repeated dose of LLP at 1.47 g/kg b.w for 90 d in female and male Wistar rats the results showed that male rat gained more body weight or grown faster than the female rat (Fig. 4A). Though the organ weight of treated and control rats in the same sex group was significantly similar (p > 0.05) (Figs. 4B–F), however, rats treated with LLP showed to have a lower organ weight when compared with control one. Generally, the organ weight is the most sensitive biomarker of drug toxicity.³⁴⁾ However, significant variations in organ weight between treated and controlled animals might occur in the absence of any morphological abnormalities.³⁷⁾ In this study, the relative organ weights of the treated rats of both sexes administered 1.47 g/kg b.w. were not statistically different from the control group suggesting that administered of effective dose of LLP was not toxic. This corroborates the finding of Tatefuji et al.,³⁶⁾ who reported that seed extract of Gnetum gnemon L. was safe for Wister rats at a dose of 1g/kg for 4 weeks with repeated dose.

Fig. 4. Effects of the Liang Leaves Powder on Body Weight and Relative Organ Weight of Rats in Sub-chronic Toxicity Study

The values represent the mean ± S.E.M. (N = 10). The serum levels of (A) AST, (B) ALT, (C) creatinine, and (D) BUN in rats. The levels of (E) WBC, (F) Total RBC, (G) HCT, (H) Hb, (I) MCV, (J) MCH, (K) MCHC and (L) Platelet count in rats. Values with same alphabet are not statistically different (p > 0.05) (n = 5).

Effect of LLP on Biochemical and Hematological Indices

Biochemical indices were conducted to appraise possible changes in the liver and renal functions of as well as the hematological parameters of rats influenced by acute and sub-chronic administration of LLP. The indices assessed for liver function are aspartate transaminase (AST) and alanine transaminase (ALT) while for renal function were BUN and creatinine (Figs. 5A–D). The results showed that there was no significant difference (p > 0.05) in AST and ALT activities in the serum of G2 rats which received 2000 mg/kg LLP (acute administration) when compared to G1 rats which received only distilled water. Similarly, there was no significant variation (p > 0.05) in the levels of BUN and creatinine in G2 rats compared to the control. The liver is the main organ responsible for metabolizing metabolites, including chemical agents and xenobiotics, and its integrity is often reflected by the levels of AST and ALT. An increased AST level is indicative of inflammation in the various organ including liver, muscle, heart, brain and red blood cells while ALT is mainly used to measure liver inflammation.³⁸⁾ When the hepatic cells are damaged, both AST and ALT will leak out of the membrane into the bloodstream,³⁹⁾ thus leading to high levels in the blood. Our observations showed that the AST and ALT levels in the treat group that received 2000 mg/kg of LLP were not significantly different from the control group. This indicates that LLP did not contain any toxic substance capable of inducing hepatic damage. Creatinine and BUN are indicators used to monitor kidney function and more specifically creatinine is a waste product left over from energy producing processes in muscles.⁴⁰⁾ An increase in the levels of both parameters in the serum indicates kidney dysfunction. The results of this study showed that the BUN and creatinine levels in the LLP-treated group were not significantly different from the control group, indicating that LLP did not cause kidney injury.

Fig. 5. Effect of Administration of Liang Leaves Powder on Clinical Biochemistry and Hematological Indices in Rats

Assessing hematological parameters is a significant and sensitive indicator, which is essential for extrapolating experimental data to clinical studies.^41,42) Evidence has demonstrated that consuming toxic plants or agents can cause changes in the hematological profile. Figures 5E–L demonstrate the impact of LLP on hematological indices of rats. It is evident that there were no significant alterations in the hematological parameters (RBC, WBC, Hb, Hct, MCV, MCHC and platelet count) investigated in the test group that received 2000 mg/kg of LLP compared to the control rats.

Biochemical study of sub-chronic administration of 1.47 g/kg b.w. of LLP to rat for 90 d shows that all biochemical parameters measured except creatinine in female rats of treated group were not significantly (p > 0.05) different from the control group of the same sex (Table 2). Serum glutamic-oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) are biochemical parameters that have been employed to predict liver damage.^38,43) This study found that SPOT was increased by 13.84%. Creatinine level in female treated rats was significantly (p > 0.05) lower than the control group. Though there was no significant sign of kidney damage, the cause of the decline in creatinine level may be due to the fluctuation of hormones with age,⁴⁰⁾ especially in female rats. The impact of LLP on the hematological indicators of treated male rats is shown in Table 3. There was no significant change found in the WBC, MCV, MCHC, or platelet counts of the rats when compared with the control. However, there was a substantially increase (p < 0.05) in RBC, Hct, and HB levels in the treated male rat compared to the control. No significant change was observed in the hematological indices investigated in the female rats (Table 3). This observation further substantiates that LLP does not contain toxic compounds capable of causing biochemical alterations.

Table 2. Effect of Administration of Liang Leaves Powder on Biochemical Parameter on Wistar Rat

Sex	Parameters	Unit	Control group (Water ad libitum)	Sub chronic toxicity group (1.97 g/kg LLP)
Male	SGOT (AST)	U/L	116.10 ± 28.84a	114.00 ± 20.23a
	SGPT (ALT)	U/L	41.40 ± 10.90a	47.13 ± 9.54a
	Blood urea nitrogen	mg/dL	21.81 ± 3.16a	22.51 ± 3.09a
	Creatinine	mg/dL	0.36 ± 0.03a	0.40 ± 0.03a
Female	SGOT (AST)	U/L	147.38 ± 43.72a	122.38 ± 43.04a
	SGPT (ALT)	U/L	47.38 ± 11.36a	46.13 ± 6.81a
	Blood urea nitrogen	mg/dL	25.08 ± 3.87a	27.51 ± 3.20a
	Creatinine	mg/dL	0.56 ± 0.10a	0.44 ± 0.05b

Remark: The values represent the mean ± S.E.M. (N = 10). Different letters indicate significant differences that compared within row (p < 0.05).

Table 3. Effect of Administration of Liang Leaves Powder on Hematological on Wistar Rat

Sex	Parameters	Unit	Control group (Water ad libitum)	Sub chronic toxicity group (1.97 g/kg LLP)
Male	WBC	x109/L	3.74 ± 0.93a	4.8 ± 2.60a
	Total RBC	x1012/L	9.48 ± 1.11b	11.79 ± 1.35a
	Hct	%	48.7 ± 5.31b	62.13 ± 6.64a
	HB	g/dL	17.35 ± 2.28b	21.64 ± 2.20a
	MCV	Fl	51.3 ± 1.57a	52.5 ± 1.60a
	MCH	Pg	18.29 ± 1.12a	18.36 ± 0.72a
	MCHC	%	35.56 ± 2.29a	35.3 ± 1.48a
	Platelet count	x1012/L	849.7 ± 205.09a	995 ± 169.88a
Female	WBC	x109/L	2.61 ± 1.07a	2.43 ± 0.63a
	Total RBC	x1012/L	11.09 ± 1.78a	11.40 ± 1.15a
	Hct	%	61.75 ± 10.50a	62.25 ± 5.60a
	HB	g/dL	21.99 ± 3.79a	21.73 ± 2.07a
	MCV	Fl	56.00 ± 1.77a	54.63 ± 1.06a
	MCH	Pg	19.79 ± 0.60a	19.19 ± 0.41a
	MCHC	%	35.69 ± 0.67a	35.00 ± 0.32a
	Platelet count	x1012/L	851.4 ± 297.1a	947.38 ± 135.8a

Remark: The values represent the mean ± S.E.M. (N = 10). Different letters indicate significant differences that compared within row (p < 0.05).

Histopathological Examination

Histological examination on acute toxicity of LLP in rat kidney and liver that received 2000 mg/kg b.w. is demonstrated in Fig. 6. It was noticed that G2 rats expressed similar liver and kidney structural cell characteristics when compared to G1 (control rats). From the renal viewing, there was no sign of nephron swelling or tubular cast noticed in the nephron likewise the glomerulus did not show any hypertrophy or proliferation of mesangial cells, podocytes and bowman capsule. Histological viewing of the liver showed that the hepatic lobule was normal with hexagon shape and the central vein in the middle of the lobule for liver cells (hepatocyte) was also normal.

Fig. 6. The Histology and Specimen of the Kidney and Liver Changed in Control Rats and Treated with 2000 mg/kg of Liang Leaves Powder

(A) Kidney specimens (B) Representation H&E-stained kidney section (C) Liver specimens (D) Representation H&E-stained liver section. In Fig. 4D, CV indicated central vein.

In addition, the histological study after 90-d administration of 1.47 g/kg LLP to rats showed normal architecture of the investigated organ with no sign of interstitial edema, cell degeneration, cytoplasmic blebbing, hyalin cast, or nucleus change in necrosis in treated rats similar to what was noticed in the control group (Fig. 7). Our data on liver-renal function indices (AST, ALT, BUN and creatinine) which reflected no alteration in the liver and renal functions is complemented by the histopathological study where no changes in the structural architecture of both organs were observed. Histopathological examination is a gold standard usually employed to evaluate the pathological changes caused by the consumption of a plant leaves on tissues and organs.³⁴⁾

Fig. 7. The Histology and Specimen of Kidney (a–d) and Liver (e–h) Showed Normal Architecture in Control Rat and Treated with 1.47 g/kg b.w. of Liang Leaves Powder

CV indicated central vein.

Acknowledgments

This research was supported by National Science, Research and Innovation Fund (NSRF) and Prince of Songkla University (Grant No. AGR6505051M).

Conflict of Interest

The authors declare no conflict of interest.

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