Phytochemical investigation on Vitex negundo leaves and their anti-inflammatory and analgesic activities

The phytochemical investigation on Vitex negundo leaves has led to the isolation of one new iridoid glucoside (8α-hydroxy-4-carboxyl-5βH-9βH-iridoid-1α- O -(6′- O -(6,7-dihydrofoliamenthonyl)-β-ᴅ-glucopyranoside, 3 ), together with three known compounds, namely agnuside ( 1 ), 6′- O - E caffeoylmussaenosidic acid ( 2 ), and 3,5-dicaffeoylquinic acid ( 4 ). The HPLC analytical study was also performed to quantify the content of agnuside ( 1 ) in dried leaves. The results indicated the very high content of 1 (3.04 ± 0.02%). The method was also validated by various parameters, including linearity (R 2 = 0.9999), precision (intra-day RSD ≤ 2.50%, inter-day RSD= 0.76%), and accuracy (recovery rates 96.58-101.86%). The animal testing data showed that the extract did not reduce pain at the doses of 9.6 and 28.8 g /kg (leaf weight/body weight) in the hot plates and pain measuring models but showed the pain reduction in the acetic acid-induced pain model. The extract at the dose of 5.6 g/kg (leaf weight/body weight) also had effects on the acute inflammation in the carrageenin-induced edema model. The extract at the dose 9.6 and 28.8 g/ kg (leaf weight/body weight) also showed significant chronic anti-inflammation, comparable to methylprednisolone at the dose 10 mg/kg on the mouse peritoneal.


INTRODUCTION
Vitex negundo L. was a shrub or small tree which was available in the plains, mountainous area and in the midland of Vietnam. It has been widely used in treatment of many diseases such as arthritis, flu, fever, cough, asthma, sprains, and colitis (National Institute of Medicinal Materials, 2006;Vu, 2007). The published chemical studies on V. negundo showed the wide spectrum of its compounds, such as iridoid glycosides, quinic acid derivatives, flavonoids, and lignans (Hu et al., 2016;Hu et al., 2017;Zheng, C.J. et al., 2015). There were also pharmacological studies on analgesic and anti-inflammatory properties of this plant (Chattopadhyay et al., 2012;Dharmasiri et al., 2003;Gill et al., 2018).
In Vietnam, so far, no study has been conducted on the compounds of this plant as well as its pharmacological activities. With the potential clinical application and consideration of available studies on this plant, the present study was conducted to give more insights into compounds of V. negundo collected in Vietnam, analytical method for quantification of main compounds, as well as its analgesic and anti-inflammatory properties in the different animal models, in order to provide firm scientific support for traditional usage, clinical application, and quality control procedure in Vietnam.

General experimental procedures
NMR experiments were conducted on a Bruker Avance III HD (400 MHz) spectrometer (Bruker)

Standard solution
Reference compound 1 was isolated in-house by various means of chromatography. The standard compound had a purity ≥ 95% as determined by HPLC and NMR. The stock standard solution was freshly prepared in methanol with concentration approximately 1.0 mg/mL. These steps of standard solution were prepared from the stock by diluting with methanol.

Sample preparation
Powdered plant material (50 mg) was extracted sonically with methanol (3.0 mL × 20 min × 3 times). After centrifugation (2800 rpm, 2 min), the supernatants were combined in a 10 mL volumetric flask. Before analysis, each sample solution was filtered through 0.4 µm membrane.

Analytical conditions
The optimal separation was determined as follows: on column Phenomenex Synergi 4u MAX-RP 80A (150×4.60 mm, 4µm) protected by a 0.2 µm guard filter (Waters) at 40 o C. The mobile phase was consisted of water containing 0.02% trifluoroformic acid (A) and acetonitrile (B). The gradient was from 12B/88A to 16B/84A in 35 min. The column was then washed with acetonitrile and re-equilibrated with initial solvent system for 15 min before the next analysis. The injected volume of sample was 10 µL, the flow rate was set at 0.6 mL/ min. The UV detector was set at 258 nm

Method validation
The method validation was conducted for various criteria, including linearity, the limits of detection (LOD) and quantification (LOQ), accuracy, and precision. The calibration curve was established with a stock solution of compound 1 (approximately 1.0 mg/mL) prepared in methanol and five further levels obtained by serially diluting with methanol (1:1). Linearity was calculated based on six concentrations and their respective HPLC peak area. The LODs and LOQs were visually determined as 3-times and 10-times signal-to-noise ratio, respectively. Peak purity was determined by 'Peak Purity' function in Agilent software with threshold set at 950. Intra-and interday precisions were evaluated on three consecutive days, each sample was examined in triplicate. The accuracy of method was evaluated by spiking three different volumes of 1 (1 mg/mL) into plant materials sample (high spike: 1000 µL; medium spike: 500 µL; low spike: 250 µL).

Statistical analysis
All samples were analyzed in triplicate. The analyzed data was expressed in the form "mean ± standard deviation (SD)". The data was calculated and processed by Microsoft Excel 2016 (Microsoft, USA).

Extract preparation for animal testing
Plant materials (5 kg) were crushed into small pieces and extracted by 70% aqueous ethanol under flux 3 times (solvent/ material ratio= 6/1). The combined extract was then evaporated under reduced pressure to two thirds of original volume and stirred for 24 hours. After that, the extract was filtered to remove settled resin and evaporated to remove completely organic solvent in order to obtain concentrated extract.

Experimental animals
Healthy mature Swiss mice (weights, 18-22 g), and mature Wistar rats (weights, 100-150 g), of both sexes were provided by the National Institute of Hygiene and Epidemiology (Hanoi, Vietnam). Animals were acclimated 5-7 days before experiments.
The experiments were carried out in the Department of Pharmacology, Hanoi Medical University, Vietnam. Animal research protocols were approved by the Ethical Council of Hanoi Medical University, Vietnam.

Hot plate test and gauge pain threshold
The mice were divided into 4 groups and administrated orally using gavage. Group I drank distilled water as the control at the dose of 0.2 mL/10g; Group II drank codeine phosphate at a dose of 20 mg/ kg; Group III, IV drank extract of V. negundo leaves at the dose of 9.6, 28.8 g/kg (leaf weight/body weight), respectively.
The heat sensitivity of mice was evaluated by hot plate test to measure the response time to temperature of mice one hour before and after the last treatment. The animals are plated on the hot plate (always maintained at 56°C) and the time until either licking or jumping occurs was recorded by stopwatch. The mice that responded too quickly (before 8 seconds) or too slowly (after 30 seconds) were then eliminated. The reaction time with heat stimulation before and after the administration of the V. negundo extracts (in seconds) was then compared among mice groups (Ankier, 1974;Vogel, 2008).
The pressure pain threshold was measured using Dynamic Plantar Aesthesiometer in which an ascending force was applied to the right paw of the mice. The distance was recorded by observing on the scale the force at which the animal felt pain (Ankier, 1974).

Acetic acid-induced writhing
White mice were divided into 4 groups each containing ten rats: group I drank distilled water as the control at the dose of 0.2 mL/10g; group II drank aspirin 150 mg/kg; group III, IV drank extract of V. negundo leaves at the dose of 9.6, 28.8 g/kg (leaf weight/body weight), respectively. The mice in the groups I, III and IV were drinking distilled water or the reagents once a day in the morning for three consecutive days. On the third day, one hour after taking the substance, 0.2 mL of acetic acid 1% was injected to mice's abdominal cavities. The number of cramping pain episodes in each mouse was counted every 5 minutes for 30 minutes after the injection of acetic acid, then was compared to the control groups (Koster, Anderson, de Beer, 1952).

Anti-inflammatory activity
The acute anti-inflammatory activity of the V. negundo leaves were evaluated in the carrageenan-induced rat paw edema model and the peritonitis rat model (Winter et al., 1962). Each model was divided into 4 groups: Group I drank distilled water as the control at the dose of 1 mL/100g; Group II drank aspirin 200 mg/kg; Group III, IV drank the V. negundo leaves at the doses of 5.6 and 16.8 g/kg/day (leaf weight/ body weight/day), respectively. Rats were given the V. negundo leaves or water or aspirin for 4 days before inducing inflammation. For the carrageenan-induced rat paw edema model, on the fourth day, one hour after taking the V. negundo leaves, 0.05 mL of 1% solution of carrageenan was injected into the right hind soles of the rat. The volume of rat paw was observed before inducing inflammation (V0), 2 hours after inducing inflammation (V2), 4 hours (V4), 6 hours (V6) and 24 hours (V24) by using a Plethysmometer.
The increase of rat paw volume was calculated by following formula V 0 : rat paw volume before inducing rat paw edema V t : rat paw volume after inducing rat paw edema The anti-inflammatory activity of the drugs was expressed as percentage inhibition (I%), which was calculated as follows: : Average increase in rat paw volume in control group : Average increase in rat paw volume in treated group In the peritonitis rat model, on the fourth day, one hour after taking the V. negundo leaves, 2 mL of solution of carrageenan (50 mg carrageenan and 1.4 ml formaldehyde mixed enough in 100ml saline solution) was injected into the peritonitis of the rat. One day after causing inflammation, inflammatory exudate was taken from the abdomen of the mouse. The number of leukocytes in 1 mL of inflammatory exudate was counted (Vogel, 2008).
The chronic anti-inflammatory effects were studied on experimental granulomas model (Vogel, 2008). In detail, chronic inflammation was induced by implanting sterile asbestos fibers weighed 6 mg which were soaked in 1% carrageenan into the skin of each mouse nape. The treatment groups included Group I drank distilled water 0.2 mL/10g; Group II drank methylprednisolone 10 mg/kg; Group III, IV drank the V. negundo leaves at the doses of 9.6, 28.8 g/kg/day (leaf weight/body weight/ day), respectively. The mice then drank distilled water or the reagents continuously for 10 days. On the eleventh day, mice were sacrificed in order to collect granuloma. Three granulomas in each group were randomly selected for microscopic pathological observation. The remaining granulomas were dried at 56°C for 18 hours then weigh after being dried. Results were expressed as percentage inhibition of granuloma in drug treated groups compared to the control group, which was calculated as follows: GT: granuloma tissue weight in treated group, GC: granuloma tissue weight in control group.

Statistical analysis
Data was shown as mean ± standard error (mean ± SE). The data was evaluated Independent Samples T-Test using SPSS program (version 18. SPSS Inc., USA). The differences were statistically significant with p < 0.05.

HPLC quantification and method validation
The optimal separation was determined as follows: on column Phenomenex Synergi 4u MAX-RP 80A (150×4.60 mm, 4µm) protected by a 0.2 µm guard filter (Waters) at 40 o C. The mobile phase was consisted of water containing 0.02% trifluoroformic acid (A) and acetonitrile (B). The gradient was from 12B/88A to 16B/84A in 35 min. The column was then washed with acetonitrile and re-equilibrated with initial solvent system for 15 min before the next analysis. The injected volume of sample was 10 µL, the flow rate was set at 0.6 mL/ min (Figure 4). The UV detector was set at 258 nm. The content of 1 in experimental sample was determined 3.04 ± 0.02% (dried weight).

Analgesic activity
The results reported in the Figure 5 showed that the extract of V. negundo leaves in both 2 doses of 9.8 g/kg/day (leaf weight/body weight/day) and 28.8 g/kg/ day (leaf weight/body weight/day) taken orally for 3 consecutive days were significantly effective in reducing a number of writhing induced by 0.2 mL acetic acid 1% at all times of study, compared to control group (p < 0.05, p < 0.01, and p < 0.001), indicating the peripheral analgesic activity of the extract. However, the Figure 6 and Figure 7 showed that the extract of V. negundo leaves showed no central analgesic activity in hot plate method and pressure pain threshold on mice with the doses of 9.6 and 28.8 g/kg/day (leaf weight/body weight/day) orally for three consecutive days.

Page 9/24
Phytochemical investigation on Vitex negundo leaves and their anti-inflammatory and analgesic activities

Acute anti-inflammatory activity
Acute anti-inflammatory effects were measured on the white rat with carrageenan-induced edema paw model. The reduced percentages of edema paw volume were presented in the Figure 8. In the control group (group 1), carrageenan injection was used to induce a local edema, which progressively increased after 2, 4, and 6 hours to 42.1, 60.8, and 69.0%, respectively. Afterwards, the edema decreased, but still remained after 24 hours. The pretreatment by administering the V. negundo leaves orally with the dose of 5.6 g/kg (leaf weight/body weight) on rat significantly decreased the paw edema levels by 10.5, 18.3, and 41.2% after 2, 4, and 6 hours, respectively.

Influence of V. negundo leaves on the number of leukocytes in inflammatory exudate
The results in the Figure 9 showed that the extract of V. negundo leaves with the dose of 16.8 g/kg (leaf weight/body weight) reduced significantly the number of leukocytes in inflammatory exudate. When compared to control group (p < 0.05), the reduction was 32.23%.

DISCUSSION
The purpose of this study was to establish a firm scientific basis for the usage of V. negundo for treatment of anti-inflammatory conditions in Vietnam. This was a necessary task because V. negundo has been widely used in Vietnamese community, however, little was known about its compounds, as well as pharmacological activity of this species cultivated in Vietnam. The phytochemical investigation on V. negundo leaves collected in Vietnam revealed that the major components are iridoid glucosides (1-3) and quinic acid (4) derivatives. Agnuside (1) was determined to be the major constituents in V. negundo leaves sample collected in Vietnam with very high content (approximately 3%). The HPLC quantification method was also validated according to ICH guideline (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2005)2005. Besides that, a new natural iridoid (3) was also isolated and structurally elucidated by combination spectroscopic and spectrometric methods (1D, 2D-NMR, HRMS).

Chronic anti-inflammatory effects of the V. negundo leaves
The results in the Figure 10 showed that the extract of V. negundo leaves with the doses of 9.6 g/kg (leaf weight/body weight), and 28.8 g/kg (leaf weight/body weight), reduced 40.20% and 41.24% granulomas weight when compared to control group, which indicated the activity against chronic inflammation (p < 0.01). (Presented as Mean ± SE (n=10). Statistical analysis: **: p < 0.01, ***: p < 0.001, T-test as compared to control) 0.001 at the dose of 50 mg/kg) (Okuyama et al., 1998). Therefore, the quantification of agnuside content showed significant values in quality control of this plant and its related herbal products. An HPLC quantification method was also developed and validated, which gave the result of content of agnuside (1) in dried leaves of V. negundo (3.04 ± 0.02%). The validation parameters were in good range for quantification (See Table II).
The extract was further examined in animal models for analgesic and anti-inflammatory activities. The V. Negundo leaves showed no statistically significant difference in response time in hot plate method at the both doses of 9.6 and 28.8 g/kg (leaf weight/body weight), indicating that the extract did not possess analgesic effect on the central nervous system in the experimental models. On the contrary, the extract did reduce the number of cramping pain attacks in the writhing mouse tests. The pain inducing experiment with acetic acid was representative in evaluating the peripheral analgesic activity, proving that the extract displayed effect on the peripheral mechanism at the experimental doses (9.6 and 28.8 g/kg). These results were in good agreement with previous pharmacological study results. Agnuside isolated from V. rotundifolia fructus showed significant writhing inhibition with oral administration at dose of 50 mg/kg (Okuyama et al., 1998). Gupta and Tandon (2005) also reported that at the dose of 500 mg/kg (extract weight/ body weight), V. negundo leaves extract was shown to delay writhing onset and significantly reduce the number of acetic acid-induced writhings (Gupta, Tandon, 2005).
As for anti-inflammatory activities, V. negundo leaves extract showed potent effects in carrageenaninduced edema rat model. The volume of rat paw edema significantly decreased by 41.16% with the dose of 5.6 g/kg (leaf weight/body weight), compared to the control group. The extract also showed the reduction in the number of leukocytes in inflammatory exudate (32.23% compared to the control group) at the dose of 16.8 g/kg (leaf weight/body weight). The results strongly suggested the extract of V. negundo leaves had activity against acute inflammatory conditions. The extract was also shown to reduce the granulomas weight (40.20 and 41.24 %) in the chronic granulomas inflammatory mouse model at the doses of 9.6 and 28.8 g/kg (leaf weight/body weight). respectively, which was comparable to methylprednisolone at the dose of 10 mg/ kg. The experiment used a carrageenan-impregnated asbestos implanted under the skin of experimental mice. The immune system of mouse would be activated but it would not be able to eliminate the inflammatory inducer, therefore causing chronic inflammatory conditions. This indicated that the extract also showed activity against chronic inflammation, and the magnitude of activity was comparable to that of methylprednisolone. Previous studies had also indicated that V. negundo extracts displayed potent anti-inflammation effects on the carrageenan-induced edema rat. Chattopadhyay and colleagues (2012) showed that the V. negundo leaf oil decreased the maximum edema to 29% at the dose of 500 µL/kg (Chattopadhyay et al., 2012). In a different case, Kulkarni (2008) reported that the 50% methanol extract of V. negundo leaves at a dose of 100 mg/kg decreased the edema to 69.08%. In a different case, Vinuchakkaravarthy's group (2011) showed that tris(2,4di-tert-butylphenyl) phosphate was isolated from the of V. negundo leaves, which reduced the raw paw edema volume significantly at the tested doses of 50 mg/kg and 70 mg/kg (Vinuchakkaravarthy et al., 2011). These results strongly indicated the effects of V. negundo leaves on the acute inflammatory conditions. However, in our experiment, there was no doseresponse effect observed and the high dose (16.8 g/kg) was not effective in the carrageenan-induced edema rat model. V. negundo constituents, such as lignans (Singh et al., 2005), glycoside steroid, and triterpenoid glycoside (Chen et al., 2014), might stimulate the immune system with the anti-cancer potential. Therefore, it was possible that the high dose (16.8 g/kg) could stimulate these immune systems. In such cases, we did not see the anti-inflammatory effects of the V. negundo. Thus, in future studies, we need to conduct more experiments with the other doses as well as study detailed molecular mechanism of action of V. negundo leaves.
There were several studies on mechanism of antiinflammatory activities of agnuside. Suksamrarn et al. (2002) reported the anti-inflammatory effects of agnuside in vitro, which revealed the mechanism of action through selective COX-2 inhibition using COX deficient murine cell lines (Suksamrarn et al., 2002) while Pandey and colleagues (2012) proved that the anti-arthritic activity of agnuside was associated with the suppression of inflammatory mediators (PGE2 and LTB4) and T-cellmediated cytokines (Th1/Th2). Moreover, agnuside was also found to inhibit vascular permeability and leukocyte migration in rat models . Other groups of compounds, such as triterpenoid, lignan, labdane and megastimane derivatives, also exhibited potent inhibitory activities on NO production in lipopolysaccharide (LPS)induced inflammation in RAW264.7 macrophages and microglial BV-2 cells (Hu et al., 2016;Li et al., 2014;Xu et al., 2019).
In conclusion, the current study provided wide range of scientific evidences to support the traditional usage and clinical application of V. negundo leaves in particular case of Vietnam, as well as a validated method for quality control for this plant and its related herbal products in the future.