Anti-inflammatory and antiviral activities of compounds from the fruit of Pouteria caimito

Abstract

Pouteria caimito is a commercially valuable tropical fruit tree and has medicinal effects in Brazilian folklore. In this thesis, thirteen compounds were isolated for the first time from the fruit of P. caimito by ethanol extraction and various chromatographic column chromatography methods. The thirteen compounds were identified by NMR, HR-ESI-MS and various physicochemical data to contain nine triterpenoids. The results of activity screening experiments showed that compounds 2, 3 and 5 exhibited anti-inflammatory activity, while most of the triterpenoids exhibited anti-RSV, and anti-HSV activity to varying degrees. Based on these results, the content of compound 5 was determined due to its good pharmacological activity to be 13.3250 mg/kg in the fresh fruit. This study scientifically demonstrated the health benefits of consuming P. caimito fruits and also provided new ideas for further development and utilization of P. caimito. It not only has good potential as an anti-inflammatory or antiviral functional food but also can be considered a natural source of triterpenoids.

Keywords:

• Pouteria caimito
• anti-inflammatory
• antiviral activity

REVIEWING EDITOR:

• M. Luisa Escudero-Gilete, Universidad de Sevilla, Spain

SUBJECTS:

• Agriculture and Food; Food Chemistry; Substitutes - Food Chemistry; Food Analysis; Nutraceuticals & Functional Foods

1. Introduction

Pouteria caimito is an evergreen fruit tree in the Sapotaceae family, native to the upper Amazon, and found in the eastern Andes (Silva et al., 2009). It is also known as Abiu, Yellow Star Apple. The fresh pulp of this fruit is translucent and sticky, with a sweet taste, most of the time it can be eaten directly and is sometimes made into a refreshing drink, ice, sherbets, and ice cream (Coronel et al., 1998). In Brazilian folk medicine, its pulp is used to relieve coughs, fevers, bronchitis, and other lung diseases (Leung & Flores, 1961). Moreover, its latex can be used not only for abscesses and diarrhea but also as a vermifuge (Lim, 2013). Many beneficial health effects of P. caimito consumption have been reported, such as extracts from seed, skin, and pulp have antimicrobial effects and inhibit acetylcholinesterase activity (Arif et al., 2022). The extracts of seed, peel, and pulp of P. caimito have acetylcholinesterase inhibitory activity and anti-microbial activity (Arif et al., 2022). Moreover, the extracts of leaves have alpha-amylase, alpha-glucosidase, and tyrosinase inhibiting activities, and also have free radical scavenging effect (Arif et al., 2022). However, in the past, most of the research subjects have focused on crude extracts and few studies have investigated what exactly are the main active components of P. caimito. This study aimed to identify the active monomeric compounds in P. caimito fruits.

Inflammation is associated with a wide range of diseases, and the literature reports that inflammation increases the risk of many chronic diseases, thereby shortening life expectancy (Jin et al., 2010). Interestingly, a review reported in 2018 suggested that our diet is also inextricably linked to inflammation, some foods could reduce the production of pro-inflammatory mediators and enhance the function of immune cells (Hosseini et al., 2018). Therefore, regular consumption of certain anti-inflammatory foods can be very beneficial to our health.

Table 1. ¹³C^a NMR Spectroscopic Data of Compounds 1 − 13 (δ in ppm).

No	1	2	3	4	5	6	7	8	9	10	11	12	13
1	–	38.2	38.6	39.6	47.1	47.1	38.0	37.3	38.2	134.4	132.3	125.2	38.9	Glc 1	105.8
2	75.6	27.0	27.0	28.3	67.1	67.2	26.6	23.6	23.4	138.7	138.0	114.5	26.0	Glc 2	75.9
3	31.6	76.9	76.9	78.4	82.3	82.3	70.2	79.9	79.9	114.9	116.1	145.2	82.5	Glc 3	78.7
4	22.5	38.4	28.4	39.6	38.9	39.1	41.4	37.3	37.5	122.5	130.3	145.8	43.5	Glc 4	71.6
5	112.3	54.9	54.8	56.1	54.8	54.8	47.2	54.5	55.2	121.2	121.4	115.9	47.8	Glc 5	78.3
6	145.9	18.2	17.1	19.1	18.0	18.1	17.5	17.8	17.7	129.9	122.5	121.3	18.4	Glc 6	62.8
7	121.8	32.7	32.8	33.5	32.6	32.9	32.3	32.0	34.8	113.2	121.0	148.8	32.5	Glc 1’	94.8
8	125.9	39.7	41.7	40.0	40.2	39.1	38.8	36.5	40.2	157.2	129.3	113.7	40.1	Glc 2’	75.7
9	146.4	46.8	47.1	48.4	46.8	46.9	47.1	47.0	49.9	111.0	113.3	165.4	48.3	Glc 3’	79.5
10	118.5	36.6	36.4	37.6	37.6	37.7	36.3	36.4	36.6	110.9	141.9	73.1	37.1	Glc 4’	71.1
11	40.2	23.2	22.9	24.0	22.9	23.0	22.1	23.3	21.2	154.4	132.8	37.2	24.0	Glc 5’	78.0
12	21.2	126.7	124.4	122.8	124.5	121.4	121.3	121.4	27.2	57.6	167.2	66.9	122.8	Glc 6’	69.1
13	37.6	138.8	138.4	145.1	138.3	144.1	144.2	144.4	41.5	132.1	151.7	69.4	144.3	Rha 1	101.5
14	33.0	41.1	41.7	42.4	41.7	40.8	41.9	41.3	49.9	122.1	111.5	71.0	42.4	Rha 2	72.2
15	37.6	28.3	27.6	28.6	27.5	27.2	27.3	25.1	30.9	143.0	109.6	35.1	28.7	Rha 3	72.6
16	24.6	25.3	23.9	24.1	23.8	22.7	23.0	23.0	24.3	132.6	157.7	173.1	23.5	Rha 4	73.9
17	37.6	46.9	46.9	46.9	47.0	45.8	45.5	37.7	48.9		56.4	64.0	47.3	Rha 5	69.9
18	32.8	53.3	52.5	42.3	52.4	41.4	40.1	41.9	15.3			30.0	42.1	Rha 6	18.9
19	37.4	71.7	38.3	46.8	38.5	45.5	46.4	46.4	16.4			18.5	46.5	Xyl 1	105.5
20	25.0	41.5	38.4	31.2	38.5	30.4	30.5	30.9	73.0			13.5	30.8	Xyl 2	74.8
21	39.5	26.5	30.3	34.5	30.2	33.4	33.0	33.8	25.2				34.1	Xyl 3	77.6
22	28.1	37.3	36.6	33.5	36.3	32.3	32.1	30.7	41.1				33.1	Xyl 4	71.1
23	22.8	28.2	28.3	29.0	28.8	28.8	64.4	27.8	22.2				64.9	Xyl 5	67.1
24	24.2	16.4	16.1	16.8	17.0	16.9	12.7	17.4	125.2				13.8
25	12.1	15.1	15.3	15.8	17.0	16.3	15.5	21.0	130.0				16.4
26	12.0	16.7	17.0	17.7	17.2	17.1	17.1	16.7	25.5				17.7
27	19.9	23.9	23.3	26.4	23.3	25.7	25.7	25.7	17.5				26.1
28	19.8	178.0	178.6	180.6	178.4	179.5	181.5	67.5	27.7				176.7
29	22.9	26.0	18.0	33.5	16.4	32.2	32.4	33.1	16.0				33.3
30		16.1	21.1	24.0	21.1	23.4	23.5	23.1	16.4				23.5
31								16.5	170.8
32									21.0

^a Measured at 100 MHz.

When we discovered that the petroleum ether and ethyl acetate fractions of P. caimito fruit possess anti-inflammatory activity, in order to find out which ingredients were at work, a series of phytochemical studies yielded the exclusion of thirteen compounds (1–13, Figure 1) including nine triterpenoids. Nitric oxide (NO) production caused by lipopolysaccharide (LPS) was effectively inhibited by compounds 2, 3, and 5 in bioassays, with IC₅₀ values ranging from 13.1 ± 0.45 to 34.5 ± 3.74 μM (Table 3). Among them, compound 5 had the strongest anti-inflammatory effect of the group with an IC₅₀ value of 13.1 ± 0.45 μM. So that the content of compound 5 in P. caimito fruit was determined by high-performance liquid chromatography (HPLC). Moreover, some compounds were evaluated for their antiviral activities by CPE reduction assay. The results showed that compounds 2, 3, 5, 9, and 13 showed anti-RSV activity with IC₅₀ values ranging from 12.5 ± 0.23 to 50.0 ± 6.25 μM (Table 4). Moreover, compounds 2, 3, 5, and 9 showed anti-HSV-1 activity with the IC₅₀ values ranging from 12.5 ± 0.63 to 50.0 ± 2.50 μM (Table 4). Herein, the isolation, structural identification, anti-inflammatory, antiviral, and antioxidant activities of these compounds are described.

Figure 1. Chemical structures of compounds 1–13. γ-tocopherol (1), 3β,19α-dihydroxy-urs-12-en-28-oic acid (2), ursolic acid (3), oleanic acid (4), 2α-hydroxy ursolic acid (5), maslinic acid (6), hederagenin (7), erythrodiol-3-acetate (8), 3-O-acetyldammarenediol-II (9), perlolyrine (10), chlorogenic acid butyl ester (11), flazine (12), loniceroside C (13).

Table 3. Inhibitory effects of compounds against LPS-induced NO production in RAW264.7 cells.

Compounds	^aIC50 (μM)	^bCC50 (μM)
Indometacin^c	14.5 ± 2.04	>200
2	34.1 ± 2.58	82.90 ± 3.52
3	34.5 ± 3.74	90.3 ± 4.33
4	>100	>100
5	13.1 ± 0.45	60.4 ± 2.92
6	^dN.A	14.7 ± 0.35
8	>100	>100
9	>100	>100
13	>100	>100

^a Half maximal inhibitory concentration and data were expressed as mean ± SD (n = 3).

^b Concentration cytotoxicity 50% and data were expressed as mean ± SD (n = 3).

^c Positive control.

^d Concentrations below CC₅₀ did not show activity.

Table 4. In vitro anti-RSV and anti-HSV-1 activities of samples.

	RSV		HSV-1
Compounds	^aIC50 (μM)	^bCC50 (μM)	^aIC50 (μM)	^bCC50 (μM)
2	25.0 ± 1.25	>100	12.5 ± 0.63	63.1 ± 0.19
3	25.0 ± 1.56	58.0 ± 0.37	25.0 ± 3.13	47.9 ± 0.51
4	>100	>100	>100	>100
5	12.5 ± 0.23	41.2 ± 0.75	12.5 ± 1.25	26.1 ± 0.16
6	^c N.A	45.8 ± 0.56	^c N.A	24.0 ± 0.79
8	>100	>100	>100	>100
9	25.0 ± 3.20	>100	50.0 ± 2.50	>100
13	50.0 ± 6.25	>100	>100	>100
Ribavirin^d	10.0 ± 2.50	>100	–	–
Acyclovir^d	–	–	1.3 ± 0.31	>100

^a Half maximal inhibitory concentration and data were expressed as mean ± SD (n = 3).

^b Concentration cytotoxicity 50% and data were expressed as mean ± SD (n = 3).

^c Concentrations below CC₅₀ did not show activity.

^d Positive control.

2. Materials and Methods

2.1. Materials

Details of the reagents and instruments involved in this thesis can be found in the supporting information.

2.2. Plant material

P. caimito fruit was collected in Taishan City, Guangdong Province in August 2020.

2.3. Isolation procedure

The extraction method was 95% ethanol extraction by percolation at room temperature. The separation method was liquid-liquid extraction followed by chromatography on silica gel column, gel column, ods, macroporous resin column and preparative high performance liquid phase combination. See supporting information for the specific operation procedure.

2.4. Activity assay

The cytotoxicities of the compounds in this study were determined by CCK-8. Anti-inflammatory activities were measured using Griess’ reagent. Antiviral efficacies were assessed by cytopathic effect (CPE). The DPPH method mentioned in our previous work was slightly modified to determine the antioxidant activity of the isolated compounds, with vitamin E as a positive control. The detailed procedure is described in the Supporting Information (Sarkar et al., 2022; Sudipta et al., 2020; Zhan et al., 2020).

3. Results

3.1. Extraction and isolation

Comparison of the physical and spectral data (Table 1) with literature revealed the compounds were γ-tocopherol (Seo et al., 2013) (1), 3β,19α-dihydroxy-urs-12-en-28-oic acid (Katsumi et al., 2021) (2), ursolic acid (Kil et al., 2019) (3), oleanic acid (Sun et al., 2004) (4), 2α-hydroxy ursolic acid (Jing et al., 2009) (5), maslinic acid (Xia et al., 2016) (6), hederagenin (Luo et al., 2006) (7), erythrodiol-3-acetate (Kim et al., 2004) (18), 3-O-acetyldammarenediol-II (Tori et al., 1988) (9), perlolyrine (Ying et al., 2013) (10), flazine (Tian et al., 2003) (11), butyl ester (Su et al., 2002) (12), loniceroside C (Kwak et al., 2003) (13).

3.2. Anti-inflammatory activity

Firstly, the effects of petroleum ether, ethyl acetate, and n-butanol fractions of P. caimito fruit were evaluated on LPS-induced NO production in RAW 264.7 cells. As shown in Table 2, the petroleum ether and n-butyl alcohol fractions inhibited LPS-induced NO production with IC₅₀ values of 26.0 ± 1.84 and 62.9 ± 4.42 μg/mL, respectively. The majority of the isolates were tested for their ability to prevent LPS-induced RAW264.7 macrophage cells from producing NO. Among them, compounds 2, 3, and 5 exhibited excellent ability to prevent LPS-induced NO production by RAW264.7 macrophages (Table 3). The cytotoxicities of compounds 2, 3, and 5 were assessed by CCK-8 assay on LPS-stimulated RAW264.7 cells. Compounds 3 and 5 exhibited cytotoxicities in LPS-stimulated RAW 264.7 cells at 12.5 µM, and compound 2 exhibited cytotoxicity in LPS-stimulated RAW 264.7 cells at 25 µM. (Figure 2A). The results revealed that compounds 2, 3, and 5 could inhibit the production of NO at a non-toxic concentration in RAW 264.7 cells (Figure 2B–D).

Figure 2. Compounds 2, 3, and 5 significantly inhibited LPS-induced NO production in RAW264.7 cells. (A) RAW264.7 cells were treated with vector (DMSO) or compounds 2, 3, and 5 (3.1, 6.3, 12.5, 25 μM) for 48 h and then cell viability was determined using the Cell Counting Kit-8 (n = 4 replicates per dose). (B-D) RAW264.7 cells were incubated with LPS (2 μg/mLmL) in the presence of compounds 2, 3, and 5. After 48 h, the content of NO in the supernatant was determined by using Griess reagent. (n = 3 per group). Data are mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 Two-way (A) or one-way (B to D) analysis of variance (ANOVA) followed by Tukey′s multiple comparison test. ns, not significant.

Table 2. Inhibitory effects of fractions against LPS-induced NO production in RAW264.7 cells.

Extracts	^aIC50 (μg/mL)	^bCC50 (μg/mL)
Petroleum ether fraction	26.0 ± 1.84	>100
Ethyl acetate fraction	>100	>100
n-Butanol fraction	62.9 ± 4.42	>100

^a Half maximal inhibitory concentration and data were expressed as mean ± SD (n = 3).

^b Concentration cytotoxicity 50% and data were expressed as mean ± SD (n = 3).

3.3. Antiviral activities

Considering the use of the fruit of P. caimito in Brazilian folklore (Leung & Flores, 1961), we performed an attempt to evaluate compounds for in vitro anti-RSV and anti-HSV-1 activities. Compounds 2, 3, 5, 9, and 13 showed anti-RSV activities with IC₅₀ values ranging from 12.5 ± 0.23 to 25.0 ± 1.56 μM (Table 4). Furthermore, compounds 2, 3, 5, and 9 showed anti-HSV-1 activities with the IC₅₀ values ranging from 12.5 ± 0.63 to 50.0 ± 2.50 μM (Table 4).

3.4. Antioxidant activity

Some isolated compounds from the fruit of P. caimito did not appear to possess significant antioxidant properties (Table 5).

Table 5. Radical scavenge potential for samples.

Compounds	^aIC50 (μM)
Vitamin E^b	23.9 ± 0.09
2	>400
3	>400
4	>400
5	>400
6	>400
8	>400
9	>400
13	>400

^a Half maximal inhibitory concentration and data were expressed as mean ± SD (n = 3).

^bPositive control.

3.5. Determination of the content of compound 5 (2α-hydroxy ursolic acid) in P. caimito fruit

Due to compound 5 (IC₅₀ = 13.1 ± 0.45 μM) showing a more potent anti-inflammatory effect than that of positive control indomethacin (IC₅₀ = 14.5 ± 2.04 μM), we measured the content of this compound in the fresh P. caimito fruit. Results of regression analysis on calibration: y = 585348x + 366374, R² = 0.9998, the linear range was 0.616 ∼ 9.240 μg. The content of compound 5 was determined in six samples and the average content of compound 5 was calculated as 13.3250 mg/kg in the fresh fruit of P. caimito. The result was shown in Table 6 (Sarkar et al., 2021).

Table 6. Contents of 2α-hydroxy ursolic acid in P. caimito fruit.

Compound	Equation	Linearity	Limit of quantification (LOQ) (μg/ml)	Limit of detection (LOD) (μg/ml)	Content(mg/kg)
2α-hydroxy ursolic acid	y = 585348x + 366374	0.9998	9.240	0.6160	13.3250 ± 0.1528^a

^a Data was expressed as mean ± SD (n = 6).

4. Discussion

In the current work, thirteen compounds including nine triterpenoids were obtained from the fruit of P. caimito. Their structures were determined by analyzing spectroscopic data (HR-ESI-MS and NMR) and comparing them with literature data. When several substances were tested for their antioxidant, antiviral, and anti-inflammatory properties, the majority of triterpenoids displayed varied degrees of anti-RSV and anti-HSV-1 activity, while compounds 2, 3, and 5 exhibited good inhibitory on LPS-induced NO production under non-toxic conditions. The overall activity screening results showed that triterpenoids are the main active components in the fruit P. caimito.

This study shifted the research object from the previous crude extract of P. caimito to its biologically active monomeric compounds. The discovery of anti-inflammatory and anti-viral ingredients has provided new ideas and directions for the further development and utilization of P. caimito. Perhaps P. caimito has great potential as an anti-inflammatory and anti-viral functional food.

Furthermore, since the total activity screening results showed that triterpenoids are the main active components in P. caimito fruits, we conducted a literature search on the isolated triterpenoids and found that triterpenoids have a variety of pharmacological effects. In addition to the anti-inflammatory and antiviral activities mentioned in this study, they also have anti-diabetic, anti-obesity, and anti-atherosclerotic effects (Banno et al., 2004; Fukushima et al., 2006; Lee & Thuong, 2010; Li et al., 2014; Miura et al., 2012; Park et al., 2002; Yu et al., 2006; Zong & Zhao, 2007). It is not only scientifically proven the health benefits of consuming P. caimito fruits but also suggested that P. caimito fruits can be considered a natural source of triterpenoids.

5. Conclusions

In summary, this paper used ethanol extraction and various column chromatography methods to isolate 13 compounds from the fruits of P. caimito for the first time, including 9 triterpenoids, their structures were ascertained by analyzing spectroscopic data and comparing them with literature data. Activity screening experiment results showed that compounds 2, 3, and 5 have anti-inflammatory activity, while most triterpenoids have varying degrees of anti-RSV and anti-HSV activity. Due to compound 5 having good pharmacological activity, the content of it in fresh fruits was determined to be 13.3250 mg/kg by HPLC. This study scientifically demonstrates the health benefits of consuming P. caimito fruits from a new perspective, and also provides new ideas for the further exploitation of P. caimito. It not only has good potential as an anti-inflammatory or antiviral functional food, but also can be considered as a natural source of triterpenoids. In the future, we can explore the differences in the content of active ingredients in P. caimito fruits from different origins and search for the best natural sources of triterpenoids.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China under Grant (Nos. 82173695, 82003743, 81972605), the Guangdong Basic and Applied Basic Research Foundation under Grant (No. 2020B1515020033, 2020A1515110076), Science and Technology Planning Project of Guangdong Province under Grant (No. 2020B121201013), Guangzhou Basic and Applied Basic Research Foundation under Grant (Nos. 202201010485, 202102080022) and the high-performance public computing service platform of Jinan University.

Notes on contributors

Guo-Cai Wang

Guo-Cai Wang is a researcher at College of Pharmacy, Jinan university, His research interests include natural products chemistry, development of new methods in isolation, structure elucidation, and target identification of bioactive natural products. material basis of medicinal efficacy of traditional Chinese medicine and research on natural active ingredients and their pharmacological activities.