Antibacterial, anti‐inflammatory, analgesic, and hemostatic activities of Acanthopanax trifoliatus (L.) merr

Abstract Acanthopanax trifoliatus (L.) Merr (AT) is a medicinal and edible plant with high nutritional value. The biological activity of A. trifoliatus (L.) Merr and its basis for injury treatment are explored in this paper. AT was ethanol‐extracted then refined separately with petroleum ether, chloroform, ethyl acetate, and n‐butanol. Active ingredients were analyzed, and anti‐bacterial, anti‐inflammatory, analgesic, and hemostatic effects were explored. Petroleum ether layer (PEL) extract produced the strongest antibacterial effect. Ethyl acetate layer (EAL) extract had the highest active substance content, with strong hemostatic and analgesic activities. Chloroform layer (CL) extract had the strongest anti‐inflammatory effect and significantly reduced IL‐1β protein expression. Active ingredients were analyzed using HPLC and UPLC‐MS to determine saponin, polyphenol, flavonoid, and characteristic ingredient contents. EAL extract had the highest polyphenol and flavonoid levels, including rutin, chlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C, which may contribute to its nutritional activities. The study provides a reliable theoretical and practical basis for the applications of AT nutraceutical products.

Flavonoids have bacteriostatic effects due to their weak alkalinity, which coagulates or deforms proteins (Tian et al., 2018). Wang Yan found that the antimicrobial effect of flavonoids derived from alcohol-elution methods was stronger than that from water, and that the polysaccharide extract showed no obvious antimicrobial effects . The total flavonoids from Acanthopanax vulgaris had the strongest bacteriostatic effect on Escherichia coli, followed by Staphylococcus aureus (Jie et al., 2016). The extracts from different parts of Thai Acanthopanax have strong in vitro antioxidant activity, and the root bark and tender leaves display the strongest free-radical scavenging activity (Sithisarn et al., 2011). Thai Acanthopanax displayed stronger DPPH free-radical scavenging activity than Acanthopanax senticosus and Acanthopanax trifoliata from Taiwan (Lee et al., 2004).
Purified flavonoid liquor from Acanthopanax vulgaris at 45 mg/ml had significantly greater antioxidant effects than that of vitamin C (Maolian et al., 2002). Hamid RA's team in Malaysia observed the inhibition rate of AT ethanol extract was 46.23% in a carrageenan-induced rat swelling inflammation model, and 45.7% in a CFA-treated rat long-term inflammation model; this effect was comparable to the anti-inflammatory effects of piroxicam and indomethacin (Hamid et al., 2013). Furthermore, Yang Huiwen found that 10, 20, and 40 mg/(kg·d) of oral flavonoid extract from AT could significantly inhibit carrageenan-induced foot swelling in rats in a dose-dependent manner (Yang et al., 2014). Our team found that AT extracts had anticancer activities in prostate cancer cells, neural cancer cells, breast cancer cells, liver cancer cells, and large cell lung cancer cells. We also provided evidence that AT terpenoids exert anticancer activities, indicating that AT may be a useful edible plant for further development as a health supplement .
AT is widely used in folk medicine in Asia for bruises, tuberculosis, ulcer healing, tinea, and physical fitness. In China, AT is commonly used for soup and can be further processed into teas (Nan, Koen, Gabriele, & Claus, 2012). In Thailand, AT is commonly known as "Phak Paem" and is used as a traditional vegetable and folk herb (Sithisarn et al., 2011). In Vietnam, AT has traditionally been used for its positive therapeutic effects, particularly as an anti-inflammatory agent (Quan et al., 2003). AT is a raw material used in winemaking and as a hot beverage in Taiwanese folk tradition (Chien et al., 2015).
It is also one of the components of traditional Malaysian Hakka tea, lei cha, due to its medicinal benefits for the common cold, jaundice, stomachache, and diarrhea. In Cambodia, Laos, and Vietnam, herbal extracts from AT bark are used to alleviate anxiety and improve memory (Muselli et al., 2015). AT has been fortified into the soaps to relieve itching and fluoride toothpaste for periodontitis treatment (Huang, 2007). It can also be used for neonatal eczema, with therapeutic effects as an antipyretic, anti-inflammatory, and detoxification aid (Min et al., 2019).
AT has been used to treat bruises, neuralgia, impotence, and gout for a long time. However, there are few pharmacological studies on the biological activities of AT. In this study, the main active components, and the antibacterial, anti-inflammatory, analgesic, and hemostatic activities of Southern Chinese AT extracts are investigated.
This research provides a reliable theoretical and practical method for studying AT, which will be of great significance for the development of functional AT products.
Diclofenac gel (1%) was purchased from Guangdong Dashenlin Pharmaceutical Co., LTD. All other chemicals used were reagent grade.
ATs are collected from different areas in the south of China, such as Enping, Yangjiang, and Shantou in Guangdong Province, Xishuangbanna in Yunnan Province, Shaowu and Longyan in Fujian Province). The six batches of ATs from Enping (20,160,315, 20,160,401, 20,160,429, 20,160,504, 20,160,601, and 20,160,618) were picked in production periods from mid-March, early April, late April, early May, early June, and mid-June, respectively.

| Preparation of extracts
Dried AT stems and leaves (600 g) were boiled in 95% ethanol (w/v) for 2 hr and then filtered to collect the ethanol extract (EAT (NBL), and water layer (WL), respectively ( Figure 1). The obtained extracts were dried at 40℃ in the vacuum, weighed, and the yield was calculated.

| Determination of saponin, polyphenol, and flavonoid content
Dry AT or extracts (equivalent to 50 mg/ml of AT) were incubated in 35% ethanol at 80℃ for 2 hr and then sonicated for 5 min. The sample solution was dissolved completely in 35% ethanol solution and diluted after filtration for further investigation. Ultraviolet spectrophotometry (Lambda 25, Perkin Elmer) was used to determine the total saponin, polyphenol, and flavonoid content. These methods were also used for content determination in samples from different regions, batches, and extracts. The total flavonoid content of the six extracts was measured as described by Cheng et al. (2017). Five milliliters of the test solution and the rutin reference solution were treated in turn with 0.3 ml of 5% NaNO 2 for 5 min, 0.3 ml of 10% Al (NO 3 ) 3 solution for 6 min, 4 ml 1 M NaOH, and finally 30% ethanol was added to make up to 10 ml solution. The absorbance was measured by UV spectrophotometry at 510 nm. The rutin standard curve was used for total flavonoid content determination.

| Anti-inflammatory activity
Anti-inflammatory effects of all extracts were determined using a TPA-induced ear edema model (García-Rodríguez et al., 2014). Male BALB/c mice (6-week-old, 4 per group) were provided by the Animal Experimental Center of SUN Yat-sen University (Guangzhou, China).
Mice were randomly divided into 14 groups, group-housed (25 ± 1℃, RH 50%), and received tap water ad libitum and a standard diet. All AT extracts were dissolved in acetone and tested at concentrations of 50 mg/ml and 100 mg/ml. For the ear edema study, both mice ears were topically treated with 20 μl vehicle (acetone) or AT extract prior to treatment with 20 µl acetone or 20 µg/ml TPA in acetone.
All mice were euthanized after 6 hr. Ear punches (6 mm diameter) were then harvested and weighed. All protocols were approved by the Institutional Animal Care and Use Committee (IACUC).
Ear samples were fixed in 10% formalin, decalcified in EDTA buffer, subjected to a series of dehydration cycles, and embedded in paraffin (Quan et al., 2003). Samples were cut to 4 μm and routinely treated using hematoxylin and eosin (H&E) staining. Histological changes were observed under a microscope.
The IL-1β ELISA assay was performed using the ear edema sam-

| Antibacterial activity
The minimal inhibitory concentration (MIC) values of EAT and the other five extracts were determined by the broth dilution method as described by Amsterdam (He et al., 2016). The McFarland standard is used for the turbidity of suspensions of micro-organisms and bacteria for the preparation of antibacterial activity (Tian et al., 2018

| Analgesic activity
The analgesic activity of AT extracts was tested on the male Wistar rats (180-220 g) using the formalin test method (Kumar et al., 2012;Hunskaar & Hole, 1987). Rats were provided by the Animal Experimental Center of Southern Medical University (Guangzhou, China) and received tap water and a standard food diet, and were also randomized for group selection. Rats were divided into formalin, control, and AT extract groups, each with 4 mice. The blank matrix was prepared using a lipophilic cream base with a mixture of polyethylene glycol 4,000 (PEG 4,000) and liquid polyethylene glycol 400 (PEG 400) at a 2:3 ratio, with 2% (mL/g) propylene glycol as the penetration enhancer. All AT extracts were separately suspended in the blank base at 5% (w/w). Diclofenac gel (DF, 1%) was used as a reference control. The right paw of the mouse was treated topically with 0.1 g blank matrix, DF, or AT by gently rubbing with the forefinger for 1 min (Mo et al., 2013). After a further 15 min, 20 μl of 10% formalin was subcutaneously injected into the right paw. The pain response was defined as the time (in seconds) spent licking and biting the injected paw. The total licking and biting time were examined during the early phase (0-5 min) and late phase (15-30 min) after formalin injection (Sen et al., 2018).

| Hemostatic activity
The Hemostatic activity was determined by measuring plasma recalcification time (Sui et al., 2009). All extracts were dissolved in 50% ethanol, filtered, and used at 0.5%, 1%, and 2% concentration. Blood samples were collected from New Zealand white rabbit ear-veins, and anticoagulated with 3.8% sodium citrate (citrate: blood = 1:9, W/W). Anticoagulant solutions were centrifuged at 3,000 r/min for 30 min, then the supernatant (plasma) was collected for testing.

| Film-form spray preparation
EAL extract (8 mg, equivalent to 18 g AT) and 5 g poly(vinyl pyrrolidone) (PVP) in anhydrous ethanol were mixed with 3 g poly (vinyl alcohol) (PVA) and purified water was added to reach a total volume of 100 ml. The solution was mixed for 30 min using a magnetic stirrer. The quality of the product was controlled by the appearance, pH value, film-forming performance, and the content determination of active ingredients. Two grams of this product was dissolved in 15 ml purified water by sonication, and the pH value was determined.
Film-forming properties, such as film-forming time, spray effect, film characteristics, and thickness, were applied. The main active ingredients were quantitatively determined according to the method in Section 2.3. The skin irritation was further studied. Twenty subjects, 10 males, and 10 females were selected with local skin trauma, such as scratches, abrasions, or finger barbs. Samples were sprayed on the wounds of subjects. The subjects' responses on odor, color, and skin irritation were recorded.

| Quantitative analysis of active components
It has been reported that A trifoliatus (L.) Merr. contains diverse active constituents of saponins and polyphenols contributing to the beneficial biological effects, including antioxidant , anti-inflammatory , and anticancer activities . Among them, the flavonoids are important material basis in polyphenols for bio-activities. Here, we established determination methods and further verified the active ingredients in different areas, batches, and extracts.
In this paper, our team tried to establish local dietary standards for this AT product. We turned to choose the simple, convenient, and feasible UV-vis spectrophotometry for fast detection of active ingredients, which is also consistent with the national standard. The The samples from Xishuangbanna and Enping contained the highest total contents of saponins, polyphenols, and flavonoids. All samples contained over 1%, 1.7%, and 1.3% of total polyphenols, total flavonoids, and total saponins, respectively. AT from Enping has been listed as a national geographical indication agricultural product with a long history of edible use and a vast area of artificial cultivation.
Here, six batches of Enping AT from March to June were chosen for further analysis. Batches 20,160,315 and 20,160,401 were fresh and tender, and were collected in mid-March and at the beginning of April. The results show that ATs were rich in saponins, polyphenols, and flavonoids, although these active ingredients decreased with the age of the vegetable. The total content of saponins, polyphenols, and flavonoids was 23-30, 30-37, and 26-41 mg/g, respectively, which indicates high nutritional value.
However, UV-vis spectrophotometry as a rough method for the determination of total components has some shortcomings here. We will also develop more accurate methods meeting with the new national standard to improve the product quality standard.

| HPLC and UPLC-MS analysis of all extracts
Plant extracts were crudely prepared with 95% ethanol, then further treated as shown Figure 1.

F I G U R E 5
The structures of chlorogenic acid, isochlorogenic acid A and Isochlorogenic acid C, respectively 3.03, 5.04, 5.61, and 5.96 min ( Figure 6). The first two peaks were consistent with the peaks for the MIX-STD, which indicates the AT extract contains chlorogenic acid and rutin, but almost no quercetin. Mass spectrometry analysis of UPLC peaks confirmed the identification of the peaks with RT of 2. 99, 4.98, 5.51, 5.97, and 7.33 min as chlorogenic acid, rutin, isochlorogenic acid A, isochlorogenic acid C, and quercetin, respectively. The identity of these compounds was based on (+)-Q-TOF high-resolution MS results (Table 1). Concerning chlorogenic acid, rutin, isochlorogenic acid A, and isochlorogenic acid C, the content of EAL extract was the highest in chlorogenic acid, and its analogues were significantly higher than that of other extract layers. The content of chlorogenic acid, rutin, isochlorogenic acid A, and isochlorogenic acid C in CL extract was lower than EAL extract, and the composition was more complex. PE extract contained the complex substance. The NBL and water layer contained only a small amount of rutin.
Biological activity is closely related to a substance's active con- analgesic effects, and no hemostatic effect. Thus, the content of chlorogenic acid and its analogues in EAL extracts was significantly higher than in other extracts and showed stronger biological activity in terms of bacteriostatic, anti-inflammatory, analgesic, and hemostatic effects, which is consistent with reports that suggest chlorogenic acid and its analogues have positive anti-inflammatory effects.
The high content of active substances in AT provides a good raw material basis for product development, while ethyl acetate extract, with good bacteriostatic, anti-inflammatory, analgesic, and hemostatic activities, has a good product development and application prospects.

| Anti-inflammatory activity
A TPA-induced ear edema model was used to evaluate the antiinflammatory activity of 50 and 100 mg/kg AT extracts. EAT, PEL, F I G U R E 6 Ultra-high liquid chromatography and mass spectrometry analysis of standard mixtures (MIX-STD) and CL significantly reduced the degree of ear swelling induced by TPA in mice at concentrations of 50 and 100 mg/kg (p < .001),

Peak UV-RT/min MS-RT/min Compound
showing positive anti-inflammatory activity. Among all extracts, CL had the strongest anti-inflammatory effect, and its inhibition rate reached almost 99.9%, even at concentrations of 50 mg/kg.
EAL also significantly reduced TPA-induced ear swelling in mice at 50 and 100 mg/kg, with inhibition rates of 47.1% and 71.9%, respectively. In addition to CL and WL, EAT, PEL, EAL, and NBL at a dose of 100 mg/kg have a higher ability to reduce ear swelling than at a dose of 50 mg/kg. NBL had its strongest activity only at 100 mg/kg concentration, while WL and NBL at 50 mg/kg had almost no anti-inflammatory effect. We next determined the histopathologic changes in the mouse ear by paraffin section and HE staining. As was shown in (Figure 7c)

| Antibacterial activity
Antimicrobial activity was studied on P.  F I G U R E 8 Analgesic effects of topical formulations of different extracts in the formalin test. Data are reported as mean ± SEM (n = 5). Differences between formalin and extract treated groups were analyzed using a Student-Newman-Keuls one-way ANOVA (*p < .001; **p < .01; ***p < .001) control group (p < .01, p < .001), and the mean values were lower than those of the control group. Therefore, the experimental results demonstrate that AT extracts have good analgesic activity.

| Hemostatic activity
AT hemostatic effects were evaluated at concentrations of 0.5%, 1.0%, and 2.0% by the plasma recalcification time (CT) of arterial blood from New Zealand white rabbits (Figure 9). Under these conditions, the shorter the plasma recalcification time was, the stronger the hemostatic activity. All extracts had stronger hemostatic activity at a concentration of 1% compared with 0.5% and 2%. The plasma recalcification time varied between groups at 1.0% concentration.
Among these, EAL extract exhibited the shortest plasma recalcifi-

| Film-form spray preparation
The ethanol crude extract of AT further refined by ethyl acetate showed the best analgesic and hemostatic activity according to our research, which is significant for wound treatment. Spray film agent is a kind of commonly used dosage form for trauma treatment. We tried to develop a stable and controllable film spraying agent of AT for wound treatment.
Our spraying agent has the characteristics of simple preparation was determined for use as the main quality control standard. Thus, the prepared spray formed a dense film around the wound, so as to prevent the entry of external bacteria and dust, and maintain a moist and healthy environment around the wound.
A skin irritation test on human subjects without a wound was carried out, and the results were satisfactory (Table 3). The skin irritation test examined the volunteers' perception of odor, color, and skin irritation. According to the feedback, the product had a slight alcoholic taste. The color of the product was light and nearly colorless. One of the subjects felt slight irritation, which may be due to individual skin discomfort caused by ethanol in the product.
Thus, the prepared spray agent has analgesic and hemostatic effects. And it is portable and fast, and offers good permeable and continuous wound care.

| CON CLUS IONS
In this study, the active ingredients in AT were analyzed and biological activities were further explored. PEL produced the most effective antibacterial activity with a lower MIC than that of other extracts.
EAL had excellent hemostatic and strong analgesic activities. CL had the strongest anti-inflammatory effects and could significantly reduce the expression of IL-1β protein in mouse ear tissue. EAL and CL extracts produced analgesic effects during the late phase, which were stronger than that of DF. Above all, the ethyl acetate layer extract had the highest content of active substances and displayed greater anti-bacterial, anti-inflammatory, analgesic, and hemostatic

Gender Odor Color Irritation
Male Ten male volunteers thought the product had a slight alcoholic taste.
Ten volunteers considered the product was colorless to light green.
Ten volunteers thought the product was not irritating.

Female
Nine volunteers thought the product had a slight alcoholic taste, one volunteer thought it was tasteless.
Ten volunteers considered the product was colorless to light green.
Nine volunteers thought it was not irritating, and one volunteer thought it was irritating.

F I G U R E 9
Determination of plasma recovery-calcium time. Data are reported as mean ± SEM (n = 5) effects, among which analgesia and hemostasis were the strongest.
Based on the component analysis of different extracts, the relationships between the substance components and pharmacodynamics were analyzed. These results provide a reliable theoretical and practical basis for the research and development of the topical application of AT products.

ACK N OWLED G M ENT
This work was supported by Allan H. Conney Laboratory for Anticancer Research, Guangdong University of Technology and Susan Lehman Cullman Laboratory for Cancer Research, Rutgers.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L A PPROVA L
Animal procedures experiments were approved by the Animal Care and Use Committee of Guangzhou University of Technology.