1. Introduction
Litsea is evergreen or a rare deciduous, dioecious tree or shrub in the family Lauraceae. There are over 400 species, mainly in tropical and subtropical Asia, but with a few species in the islands of the Pacific, Australia, and North and Central America, and 27 species in Thailand [
1,
2]. The plant in
Litsea species has been used as traditional herbal medicines for thousands of years [
3]. Twenty plants of the genus
Litsea are found to be important traditional medicines in China for treating diarrhea, stomachache, dyspepsia, gastroenteritis, diabetes, edema, cold, arthritis, asthma, pain, traumatic injury, etc. [
4]. One of the
Litsea plants,
Litsea cubeba, different parts of this plant, such as bark, leaf, root, and fruits, are used for treating many kinds of diseases [
2]. This plant also exhibited antimicrobial, antioxidant, anti-cancer, anti-inflammatory, anti-diabetic, anti-insecticidal, and hepatoprotective activities [
2].
Litsea martabanica (Kurz) Hook.f. is one of the species found in Thailand and is also distributed in China and Myanmar (
Figure 1) [
1]. The history of utilization of this plant is based on the wisdom of the highland communities. Various parts, i.e., the roots, leaves, and stems, have been traditionally used as medicine in the highland area of the northern part of Thailand for curing kidney disease, curing toxic allergy symptoms, and detoxification [
5]. Detoxification or removal of toxins in humankind is an alternative way to promote good health for the people in the highland area who use pesticides and insecticides in daily life. The accumulation of pesticides in the body may be through the consumption of contaminated food or exposure in the occupational environment [
6]. Organophosphate and carbamate are commonly used pesticides due to a short half-life and are non-persistent in the environment [
7]. These pesticides cause acetylcholinesterase (AChE) enzyme inhibition, leading to an increase of acetylcholine (ACh) at the synapses and neuromuscular junctions. Organophosphates (OPs) are irreversible AChE inhibitors [
8]. AChE inhibition causes muscarinic and nicotinic toxicity, including cramps, increased salivation, lacrimation, muscular weakness, paralysis, muscular fasciculation, diarrhea, and blurred vision [
9]. It has been reported that OP pesticides induced reactive oxygen species (ROS) generation, which alters an antioxidant system leading oxidative damage to the cells [
10]. Humans with occupational exposes to the pesticide have been reported to have increased lipid peroxidation and significantly reduced AChE activity [
11,
12]. The levels of antioxidant enzymes, catalase, superoxide dismutase, glutathione peroxidase, and glutathione reductase, as well as non-enzymatic antioxidant, reduced glutathione, were changed in organophosphate poisoning [
13,
14]. Prolonged exposure to these pesticides was reported to be associated with various types of cancers [
15] and pathologic liver diseases such as hepatitis, fibrosis, and cirrhosis [
16,
17].
The liver is the first organ to eliminate potentially harmful xenobiotics and pesticides through the cytochrome P450 enzyme metabolism. However, a high dose of pesticide exposure may reduce the detoxifying function of the liver, leading to liver cell injury. The pesticide-induced liver toxicity involves ROS generation, which causes oxidative damage to the liver cells. Some reports suggested that antioxidant substances may be beneficial in the treatment of acute organophosphate pesticide poisoning [
18,
19]. The generation of oxygen-free radicals during pesticide exposure can be neutralized by various enzymatic and non-enzymatic antioxidant systems [
20,
21]. Plants are considered as an important source to meliorate ROS. The non-enzymatic antioxidants, including ascorbic acid, glutathione, proline, carotenoids, phenolic acids, flavonoids, tannins, etc., were found in many plants [
22]. Some research findings purposed the potential use of many non-enzymatic antioxidant substances to eliminate the adverse effect of pesticides [
23]. An in vitro experimental study has reported that vitamin C and vitamin E may ameliorate oxidative stress induced by organophosphate pesticides through the decreasing of lipid peroxidation in erythrocytes [
24,
25]. A study in Wistar rats has demonstrated that vitamin C pretreatment improves sensorimotor and cognitive functions in acute short-term chlorpyrifos-exposed rats [
26].
Besides indigenous knowledge, there are no scientific data, especially pharmacological activities, to support the traditional use of
L. martabanica as a detoxifying agent. In the present study, we investigated the anti-pesticide potential of
L. martabanica extract on rats. AChE activity, which represents pesticide exposure, was measured in the rats’ blood samples. Other parameters, including body weight change, blood hematology, blood chemistry profiles, and internal organ weight, were determined. Many plants in the
Litsea species have been reported to possess high antioxidant activity [
2,
3]. Therefore, we investigated the in vitro antioxidant activity of
L. martabanica extract as well.
3. Discussion
The root of L. martabanica was traditionally used for detoxification by the highland communities in Thailand. However, scientific data to support the traditional use of this plant are still insufficient. In the present study, we investigated the microscopic character of L. martabanica, as well as the chemical patterns, antioxidant activity, and anti-pesticide potential of L. martabanica root extract.
Antioxidants are essential substances to scavenge free radicals and prevent oxidative damage to the cells. The single antioxidant method is insufficient to study the antioxidant capacity of the plant samples. The measurement of antioxidant activity needs to use the various models to evaluate antioxidant mechanisms [
29]. DPPH assay, superoxide radical assay, ABTS assay, FRAP assay, and total phenolic content are the methods used for screening the antioxidant activity of plant samples [
30,
31]. The results of all antioxidant assays indicated that
L. martabanica root extract exhibited high antioxidant activities. Various plants in the genus Litsea provided a rich source of natural antioxidants to possess free radical scavenging potential [
3].
L. martabanica extracts and their fractions demonstrated different antioxidant efficacy in each assay model. These results correlated with the previous scientific reports. For example,
L. coreana var. lanuginose or Hawk primary leaf tea infusion (HPI) had high polyphenols contents and exhibited scavenging activity in DPPH and FRAP assay [
32]. The methanol extract of
L. cubeba showed remarkable antioxidant activity in DPPH assay, peroxidase/guaiacol assay, and thiobarbituric acid (TBA) test in comparison with α-tocopherol and ascorbic acid [
33]. The study of chemical constituents of 20
litsea plant species in China has been reported compose of flavonoids, terpenoids, alkaloids, butanolides and butenolactones, lignans, amides, steroids, fatty acids, and megastigmanes [
4,
34].
Flavonoids and terpenoids are important bioactive constituents in this genus and exert a therapeutic effect in preventing or slowing oxidative stress-related diseases [
4]. Phenolic compounds are considered secondary metabolites synthesized by plants. These compounds play an essential role in multiple biological effects, including antioxidant activity by scavenging free radicals [
35,
36,
37,
38]. The results of chemical composition in this study showed the presence of phenolics, flavonoids, and terpenes in the root of
L. martabanica. The study in TPC revealed that the extracts and their fractions contain various amounts of total phenolics. Therefore, the high antioxidant activity of
L. martabanica may result from the presence of these compounds. Antioxidant activity study in the plant extract represents an important role. Since substances with low antioxidant activity in vitro will probably show little efficacy in vivo [
39].
L. martabanica extracts and their fractions exhibited high antioxidant properties. Therefore, we selected crude water extract to investigate the anti-pesticide potential in rats, since this part was utilized for detoxification purposes by people in the highland community.
Chlorpyrifos was used to study the effect of
L. martabanica on pesticide-exposed rats. The results showed a decrease in AChE activity in the chlorpyrifos-treated group. However, treatment with
L. martabanica extract tended to restore AChE activity, especially at the high doses of 750 and 250 mg/kg. From this result,
L. martabanica extract may potentially be used as an anti-pesticide agent. Organophosphate pesticide toxicity is mainly due to AChE inhibition, which causes acetylcholine accumulation. Other mechanisms are involved in oxidative stress and free radical generation [
40,
41]. Oxidative stress induction by pesticides may occur in many ways [
6]. The central mechanism results from the autoxidation process, which increases reactive oxygen species (ROS) production. Some pesticides can alter electron transport chains in mitochondria and endoplasmic reticulum, leading to ROS overproduction. Moreover, pesticides can also inhibit antioxidant and associated enzymes or inhibit the biosynthesis of antioxidants such as glutathione [
6]. It has been reported that antioxidant enzymes, such as superoxide dismutase (SOD), catalase, and glutathione-S-transferase activities, are decreased in chlorpyrifos intoxication [
42].
The flavonoids found in many plants are powerful natural substances to scavenge free radicals [
36,
38]. The antioxidant activity of flavonoids is reported to correlate with polyphenolic structures [
43,
44]. In our study, the crude water extract consists of phenolic compounds and flavonoids. Therefore, high antioxidant activities may relate to their structure. We suggest that the anti-pesticide potential of
L. martabanica extract may be partly due to antioxidant properties. It has been reported that acute oral poisoning by chlorpyrifos involves AChE inhibition [
45]. In this study, chlorpyrifos-exposed rats showed signs of toxicity, such as piloerection, irregular respiratory patterns, and isolation from the group. The rats treated with
L. martabanica extract exhibited behavior similar to the normal group. This effect may result from the anti-pesticide potential of
L. martabanica extract, which could increase AChE activity.
The hematological analysis is the sensitive indicator to assess the toxicity of the plant extract, since the ingestion of toxic compounds could change various parameters in the hematological system [
46]. RBC, white blood cell (WBC), and platelets are used to assess the health of laboratory animals. RBC function involves carrying oxygen from the lungs to the body as well as bringing carbon dioxide back to the lungs. RBCs volume decreased in various conditions, including blood loss, immune-mediated hemolysis, inflammatory disease, renal disease, iron deficiency, myelodysplastic disease, genetic disorders, and neoplasia [
47]. WBCs serve to eliminate foreign bodies. Therefore, WBC counts may be used to indicate infection, inflammation, and immune system disorders. PLTs are cells that play an important role in blood clotting. A decreased number of platelets or thrombocytopenia is associated with bleeding [
47]. The MCV, MCH, and MCHC represent the size of RBC, the amount of hemoglobin in RBC, and the concentration of hemoglobin in an average RBC, respectively [
48]. In this study, the values of MCV, MCH, and MCHC increased in the pesticide-exposed rats. This effect may be compensated by increasing the RBC volume to carry the oxygen supply to the rats’ bodies. Our results correlate with a previous study, in which short-term chlorpyrifos exposure caused a decrease of RBCs and HCT and an increase of MCV, MCH, and MCHC in rats [
49,
50] However, abnormal hematological parameters could be normalized by treatment with
L. martabanica extract.
Blood chemistry profiles were measured to assess the physiological and pathology state of vital organs of laboratory animals. The kidney function of rats was assessed by BUN and Cr measurement. The results showed a slight increase in BUN and Cr levels in chlorpyrifos-exposed rats.
L. martabanica treatment tended to reduce BUN and Cr levels, and these values did not differ from the normal group. Release of AST, ALT, and ALP into the blood occurred after liver cells injury. Therefore, the increase in these enzymes indicated liver cell damage. Bilirubin (TB and DB) is an indication of the detoxification function of the liver. An increase in TB and DB in the bloodstream demonstrated abnormal liver function in the detoxification of toxic substances. The levels of TP and ALB reflect the synthetic function of the liver. In chronic liver diseases, the liver loses the synthetic function, which causes a reduction in TP and ALB levels in the bloodstream. ALB also decreased in renal diseases due to loss from the glomerulus [
51,
52]. Chlorpyrifos-induced toxicity in rats involved in free radical generation leading to liver cell damage and increased liver enzymes [
40,
41]. In this study, the liver biomarkers TB, DB, AST, ALT, and ALP increased in chlorpyrifos-exposed rats. These results correlate with previous research [
40,
41,
53].
L. martabanica extract treatment may protect the liver cell, as the results revealed the reduction of abnormal values of liver biomarkers. All clinical chemistry values of rats treated with the extracts comparable with those of the normal group. The results showed no aberration to indicate the presence of a physiological abnormality in the rats and the pathology of the vital organs such as the liver and kidneys. There was little difference in elevation or decline in some clinical chemistry values, which were not affected in liver and kidney function, and all values were within the reference ranges [
28].
The liver histology results revealed the necrosis of hepatic cells in the chlorpyrifos-exposed group with an increasing number of sinusoids dilatation. The results correlate with those previously reported by Albasher and colleagues [
54].
L. martabanica extract treatment helped to protect the liver cells from damage in the rats. The histopathology results showed a reduced number of sinusoid dilation and no hepatic necrosis in the extract-treated group. The liver cells of rats in the treatment group varied in shapes and sizes and exhibited vesicles with small nuclei. These are signs of hepatic regeneration that cause the restoration of the total number and mass of hepatocytes. Loss of liver mass can be induced by toxic chemicals administration. This process is followed by an inflammatory response and a regeneration response [
55]. We suggest that the
L. martabanica extract may improve liver function and protect against oxidative damage induced by chlorpyrifos.
4. Materials and Methods
4.1. Plant Material
Litsea martabanica was collected from Chiang Mai province, Thailand. The plant material was identified by the taxonomist. The voucher specimen was deposited in the Queen Sirikit Botanical Garden (No. WP 7185). The roots of
L. martabanica were selected, reduced in size and dried in the hot air oven until the moisture was less than 10%, after which they were pulverized. The powder of the plant material was evaluated for their quality of raw material following the methods described in the Thai Herbal Pharmacopoeia 2018 [
27].
4.2. Extraction of L. martabanica (Root)
The extraction process followed traditional methods. The coarse powder of the roots was extracted by decoction using water as a solvent. The extract was filtrated, concentrated until % total soluble solid or Brix = 3, and then dried by a spray dryer. Besides the water extract, the root of L. martabanica was extracted with 95% ethanol. The crude ethanol extract was separated by partition technique using n-hexane and chloroform (CHCl3), respectively. The fractions of n-hexane, CHCl3, and aqueous ethanol were evaporated and used for in vitro antioxidant activity study.
4.3. Chemical Profile by High Performance Thin Layer Chromatography
The extract samples (1 mg) were separately dissolved in 1 mL of aqueous ethanol as a test solution. Standards (apigenin, caffeic acid, gallic acid, kaemferol, pinene, and quercetin) were each prepared in the concentration of 1 mg/1 mL. A CAMAG (Muttenz, Switzerland) HPTLC system, comprising a Linomat 5 automatic applicator with a 10 mL syringe, CAMAG automatic developing Chamber 2 (ADC 2), Camag TLC scanner 4, and winCATS software version 1.4 was used. For HPTLC fingerprinting analysis, 2 μL of the test solution and 2 μL of the standard solution were loaded as 8 mm band length in the Silica Gel GF254 TLC plate. The plate was kept in TLC twin trough developing chamber (after saturated with solvent vapor) with the mobile phase (Ethyl acetate: Methanol:Water = 70:26:4). (Ethyl acetate: Methanol:Water = 70:26:4). Densitometric scanning was performed with a TLC scanner equipped with winCATS software. The plate was scanned at 254, 280, and 320 nm. The plate was kept in a photo-documentation chamber (CAMAG TLC Visualizer 2) and captured the images at White light, UV 254 nm, and UV 366 nm. The developed plate was sprayed with respective spray reagents (anisaldehyde, DPPH, natural product spraying reagent) and dried at 100 °C in a hot air oven.
4.4. DPPH Assay
The DPPH assay was performed to evaluate the free radical scavenging activity of the extract fractions. The DPPH (2,2-diphenyl-1-picrylhydrazyl) was dissolved in methanol at a final concentration of 80 µg/mL [
56]. The extracts were diluted in various concentrations. The assay method was done on a 96-wells plate as described by Phull and co-workers [
57]. Each diluted extract (20 µL) was pipetted into a separate well. Then, DPPH solution (180 μL) was added and mixed. The plate was incubated at room temperature for 30 min in the dark. The absorbance was measured at 517 nm using a microplate reader. Gallic acid and methanol were used as a reference standard and control, respectively. The percentage of DPPH scavenging activity was calculated using the formula as Equation (1):
The concentration of the sample required for the inhibition of 50% of DPPH radicals was expressed as IC
50 values [
56]. The IC
50 values were calculated using linear regression analysis and used to indicate the antioxidant capacity of the extract.
4.5. Superoxide Radical Assay
The assay was performed to assess the antioxidant activity of the test sample in scavenging superoxide free radicals. Phenazine methosulfate (PMS) and nicotinamide adenine dinucleotide (NADH) were used to generate superoxide free radicals in the system. Then, superoxide radicals reduced nitro blue tetrazolium (NBT) to purple formazan [
58,
59]. The reagents PMS (25 μM), NADH (0.5 mM), and NBT (0.2 mM) were dissolved in phosphate buffer solution (pH 7.4). To perform the assay, NBT solution (50 μL), NADH solution (50 μL), and different concentrations of samples (50 μL) were pipetted into a 96-well plate and mixed. Then, PMS solution (50 μL) was added to the well. The plate was mixed and sat at room temperature for 10 min. Then measured the OD at 560 nm using a microplate reader. Gallic acid and phosphate buffer solution were used as a reference standard and control, respectively. The percentage of superoxide radicals scavenging and the IC
50 values were calculated by the same equation as the DPPH assay.
4.6. 2,2′-Azino-Bis-(3-Ethylbenzothiazoline-6-Sulfonic Acid) (ABTS) Assay
ABTS radical scavenging activity of the extracts was conducted according to method described by Sharopov and co-workers [
60]. The ABTS reagent was prepared by dissolving 38 mg ABTS reagent in 10 mL deionized purified water (final concentration was 7.0 mM). Then, 6.5 mg potassium persulfate was added to the ABTS solution and allowed to react for 16 h to form the stable ABTS
•+ radical cation. After 16 h of incubation, ABTS solution was diluted with distilled water to obtain a final absorbance value between 0.700 ± 0.02 at 630 nm. To perform the ABTS assay, 10 µL of diluted extracts were loaded into a 96-well plate, and 190 µL of ABTS reagent was added to the well. The absorbance was measured at 630 nm after 15 min of mixture reaction. Trolox was used as standard substance. The results were expressed in milligram equivalents of trolox per gram of dry weight extract.
4.7. Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP assay was conducted according to the FRAP assay method with slight modifications [
61,
62]. FRAP reagent was prepared freshly by mixing 300 mM acetate buffer pH 3.6, 10 mM TPTZ (2,4,6-tri(2-pyridyl)-s-triazine) in 40 mM HCl, and 20 mM FeCl
3·6H
2O in a volume ratio 10:1:1. The FRAP working solution was warmed at 37 °C for 30 min prior to the assay. For the determination of the FRAP assay, 10 μL of the diluted test compound was mixed with 190 μL FRAP reagent in a 96-well plate, left for 5 min at room temperature, and the absorbance was measured at 595 nm in a microplate reader [
60]. Ferrous sulphate (FeSO
4) was used to generate the standard curve. FRAP values were expressed as mM Fe (II)/g dry weight extract.
4.8. Total Phenolics Content
Total phenolics content of the extracts was determined using Folin-Ciocalteu method [
63] with slight modifications. The test sample (10 μL) of extract diluted appropriately in dimethyl sulfoxide (DMSO) was mixed with 100 μL Folin-Ciocalteu’s phenol reagent freshly diluted 1/10 with distilled water. After five minutes of incubation, 100 μL of 7.5% Na
2CO
3 solution was added, and left for 60 min, before measurement of absorbance at 650 nm in a microplate reader. Appropriate blanks (DMSO) and standard (gallic acid in DMSO) were run simultaneously. The phenolic content was calculated as gallic acid equivalents (GAE mg/g dry weight extract) on the basis of a standard curve of gallic acid [
64].
4.9. Anti-Pesticide Potential
4.9.1. Animals
Male Sprague-Dawley rats, weighing 180–200 g, were detained from the National Laboratory Animal Center, Nakorn Pathom. They were housed under standard environmental conditions of temperature at 24 ± 1 °C under a 12 h dark-light cycle. All animals had free access to drinking water and standard pellet diet (082 C.P. MICE FEED, S.W.T. Co., Ltd., Samut Prakan, Thailand). They were acclimatized at least one week before starting the experiments. The Animal Ethics Committee of Faculty of Medicine, Chiang Mai University approved all experimental protocols, No. 49/2559.
4.9.2. Experimental Groups
The anti-pesticide potential of
L. martabanica water extract was modified from the method previously reported [
65]. Male rats were divided into five groups of six animals each.
Group 1, normal group: rats received no treatment, only 2 mL/kg of distilled water by gavage daily for 16 days and were used to determine the normal values of tested parameters.
Group 2, control group: rats received 2 mL/kg of distilled water by gavage daily for 16 days (four rounds).
Group 3, test group: rats received the cycle dose of the root water extract of L. martabanica 7.5 mg/kg for 2 days, then 2.5 mg/kg for 2 days; each rat received the extract daily for 16 days (four rounds).
Group 4, test group: rats received the cycle dose of the root water extract of L. martabanica 75 mg/kg for 2 days, then 25 mg/kg for 2 days; each rat received the extract daily for 16 days (four rounds).
Group 5, test group: rats received the cycle dose of the root water extract of L. martabanica 750 mg/kg for 2 days, then 250 mg/kg for 2 days; each rat received the extract daily for 16 days (four rounds).
The rats in group 3 to 5 received the extract in a way that mimics the traditional methods of tribal communities on the highlands. Distilled water and L. martabanica extract were orally given to the rats 30 min prior to receiving chlorpyrifos (Sigma) at a dose of 16 mg/kg. On the 17th day, all rats were anesthetized with phenobarbital sodium (50 mg/kg, intraperitoneally). A cannula was inserted into the common carotid artery for blood collection. A blood sample of each rat was distributed into a clean tube without anticoagulant and a tube with anticoagulant (EDTA).
4.10. Assay of AChE Activity
AChE activity was determined by using an AChE assay kit according to the assay protocols (Sigma) [
66]. Briefly, whole blood samples were diluted (1:40) with assay buffer, pH 7.5. Then, 10 µL of samples was transferred into separate wells of the 96-well plate and 190 mL of the working reagent were added to all samples. The reaction mixtures were mixed and incubated at room temperature. The absorbance was monitored at 2 min and 10 min, respectively, by a microplate reader at 412 nm. AChE activity was calculated using the formula as Equation (2):
A = absorbance; 200 = equivalent activity (units/L) of the calibrator when assayed is read at 2 and 10 min; n = dilution factor
4.11. Observation of Behavioral Change and Toxicological Signs
Behavior change after chlorpyrifos and
L. martabanica extracts administration were observed in the rats. The signs of toxicity, such as piloerection, diarrhea, tremor, lack of coordination, salivation, lacrimation, and others, were observed and recorded [
65].
4.12. Body Weight Change, Internal Organ Weight, and Histopathological Studies
During the experiment, the rats’ body weight was measured once daily. On day 17, the rats were sacrificed and the liver removed for weighing and gross pathological detection. The liver was preserved in 10% neutral buffered formaldehyde solution for histopathological examination.
4.13. Hematology Analysis
Blood samples were collected and determined, and blood count was completed using the automatic hematology system to evaluate red blood cell (RBC), white blood cell (WBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT), neutrophil (Nu), lymphocyte (lymph), monocyte (Mono), eosinophil (E), and basophil (Ba).
4.14. Blood Chemistry Analysis
Clotted blood samples were centrifuged to collect the serum. Blood chemistry, such as blood urea nitrogen (BUN), creatinine (Cr), total protein (TP), albumin (ALB), total bilirubin (TB), direct bilirubin (DB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP), was analyzed.
4.15. Statistical Analysis
For in vitro antioxidant assays, data were presented as the mean ± standard error of the mean (S.E.M) from three independent experiments. For in vivo experiments, statistical comparisons between the mean of each group were analyzed using the one-way ANOVA with Post Hoc multiple comparison. A value of p < 0.05 was considered statistically significant.