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Original article
01 2021
:15;
103503
doi:
10.1016/j.arabjc.2021.103503

Comparative analysis of phytochemical profile, antioxidant and anti-inflammatory activity from Hibiscus manihot L. flower

, , ,
a School of Pharmaceutical Science, Zhejiang Chinese Medical University, Hangzhou 311402, PR China
b Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China
c College of Chemistry, Chemical Engineering and Resource Utilization, Northeast Forestry University, Harbin 150040, PR China

⁎Corresponding author. zcmucq@163.com (Qi Cui)

Received: , Accepted: ,
© The Author(s) 2021
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
1 These authors contributed equally to this work.

Abstract

Hibiscus manihot L. is a kind of healthy plant with edible value and health benefits, which possesses multiple pharmacological activities that are closely related to antioxidant and anti-inflammatory activities. The dynamic changes of main active components and biological activities in Hibiscus manihot L. flower (HMLF) during its flowering period were systematically studied to determine the appropriate harvest time. Chemopreventive efficacies of the investigated HMLF extracts, by means of their anti-inflammatory and antioxidant activities, were assessed. The sample harvested on early August had the supreme total flavonoid content, total phenolic content and the strongest antioxidant activity (DPPH radical scavenging activity (IC50 0.160 mg/mL), ABTS radical scavenging activity (1.570 mmol/g Trolox), reducing power (IC50 0.101 mg/mL) and FRAP (3.644 mmol FeSO4/g)). The results of principal component analysis indicated that the primary active components included hyperin, isoquercetin, hibifolin and quercetin-3′-O-glucoside, which were strongly associated with the antioxidant activity in the early August sample, while neochlorogenic acid, chlorogenic acid and caffeic acid were associated with the anti-inflammatory activity. The extracts significantly inhibited lipopolysaccharide-induced nitric oxide production in RAW264.7 cells, especially the samples harvested around August, which was only 3.569 μΜ with the inhibition ratio of>50%. This study indicated that HMLF harvested on the early August possessed the highest antioxidant and anti-inflammatory potential and could be used as high bioactive resources for healthy production.

Keywords

Hibiscus manihot L.
Harvest time
Antioxidant activity
Anti-inflammatory activity
Phenolic compounds
1

1 Introduction

Oxidative stress and inflammation were considered to be the central mechanisms involved in various disease pathologies. Several studies have provided scientific evidence about the interlinking pathways governing oxidative stress-induced inflammation and vice versa (Means et al., 2017; Shingnaisui et al., 2018). Inflammatory cells release reactive oxygen species (ROS) at the site of inflammation leading to exaggerated oxidative damage. In addition, ROS and oxidative stress products enhance pro-inflammatory responses (Li et al., 2018; McGarry et al., 2018). Traditional knowledge on medicinal plants in nature and its active molecules were always the favorite choice of researchers to develop modern drugs. With minimal or no toxicity, medicinal plants have found utmost priority for screening and developing new therapeutics against different kinds of diseases. Therefore, natural sources of antioxidants and anti-inflammatory agents have become the focus of attention, such as polyphenols extracts (Shahidi and Ambigaipalan, 2015; Li et al., 2019; Duthie et al., 2000).

Hibiscus manihot L. is Malvaceae therophyte, which has been identified as genocide by biology during the national agricultural resources survey in 1984 (Lu and Jia, 2015). However, in August 2003, this plant was discovered through the efforts of many experts in Hebei Xingtai area. Now there are still no records in the “Chinese pharmacopoeia” and the ministry of health and drug standards promulgated. Hibiscus manihot L. flowers have been used as health food in the market, such as tea, which is conductive to the expansion of blood vessels and is the favorite choice for patients with cardiovascular and cerebrovascular diseases. Flavonoids are the main bioactive compounds in H. manihot L. flower (HMLF), which accumulates in dozens of fold higher concentrations than those in other flavonoids rich plant species such as ginkgo and soybeans (Wei et al., 2012). Chemical studies revealed that flavonoids and phenolic acids represented the main active polyphenols in HMLF. Rutin, hyperin, isoquercetin, hibifolin, myricetin, quercetin-3'-O-glucoside and quercetin were found to be the dominating flavone glycoside and flavone, which possessed pharmacological activities including antioxidant, anti-inflammatory, antibacterial, anticonvulsant, cardioprotective and neuroprotective actions (Ai et al., 2013; Zhou, 2012; Mozhgan and Kamran, 2020). As representative phenolic acid in HMLF, chlorogenic acid was widely distributed in plants and exhibited antioxidant, anti-inflammatory, anti-hypertension, antidiabetic and anti-obesity properties (Hee et al., 2017; Francisco et al., 2013; Cho et al., 2010; Jin et al., 2015; Muhammad et al., 2018). Hyperin was a major pharmacologically active compound found in HMLF, which possessed multiple bioactivities including anticancer, antioxidant, anti-inflammatory, cardioprotective and antispasmodic activities (Patel et al., 2018). All the information suggested that H. manihot L. is a plant of high pharmacological activity and research value.

The variations in active phytochemicals are affected by multi-factor, such as genotypes, cultivation techniques, harvesting time and climatic conditions (Wei et al., 2013). Scavo et al., 2019 reported that climatic conditions and harvest time had a marked influence on the presence of sesquiterpene lactones in Cynara cardunculus L. Significant differences were also displayed on the seasonal variations in the concentrations of chemical compounds of Echinacea purpurea and Echinacea pallida (Thomsen et al., 2012). Shan et al., 2019 confirmed the appropriate harvest time for quantitative analysis and bioactivity of Gardenia jasminoides fruits. Therefore, the phytochemicals in plants may vary at different harvest time, leading to the variation in bioactivity, which can provide the useful information for determining the desirable harvest time.

This study we present the variations in the chemical compositions, as well as total phenolic and total flavonoid contents, antioxidant activities (DPPH radical scavenging activity, ABTS radical scavenging activity, reducing power and FRAP) and anti-inflammatory activity of H. manihot L. flower extracts at different harvest time. Principal component analysis (PCA) was carried out to detect cluster formation and investigate the relationships among samples of different harvest time. To our best knowledge, no research on simultaneous determination of the antioxidant and anti-inflammatory activities of HMLF extracts has ever been conducted.

2

2 Materials and methods

2.1

2.1 Plant materials and extraction

Hibiscus manihot L. flower (HMLF) harvested in the city of Harbin (Heilongjiang Province, China, northern latitude 45°43′9″, east longitude 126°38′5″) for every seven days throughout the flowering period. HMLF were cleaned and dried in the shade at room temperature. The dried HMLF were powdered and extracted thrice with 60% ethanol for 45 min in an ultrasonic bath. Then filtered and stored at 4 ℃ until use.

2.2

2.2 Chemicals

Neochlorogenic acid (NA), chlorogenic acid (CHA), caffeic acid (CAA), rutin (RU), hyperin (HY), isoquercetin (ISQ), hibifolin (HI), myricetin (MY), quercetin-3′-O-glucoside (QOG), quercetin (QU), ascorbic acid (Vc), butylated hydroxytoluene (BHT), 2,2-diphenyl-2-picrylhydrazyl (DPPH), Folin-Ciocalteu (FC) reagent, DMSO, lipopolysaccharide (LPS) and MTT were all ordered from Sigma-Aldrich. The total antioxidant capacity assay kits for ABTS and FRAP assays were obtained from Beyotime Institute of Biotechnology, China. ACN and H3PO4 used in section 2.3 were acquired from J&K Chemicals Ltd. All other reagents and chemicals were purchased from Tianjin Fuyu Fine Chemical Co. Ltd.

2.3

2.3 Determination of HMLF by HPLC

Ten target components NA, CHA, CAA, RU, HY, ISQ, HI, MY, QOG and QU were simultaneously determined by the same equipment as described by Cui et al., 2020. At 1 mL/min and 30 ℃, the elution sequence for separation of ten target compounds with 0.5% H3PO4 solution (A) and ACN (B) was as follows: 90–83% A (0–40 min), 83–67% A (40–59 min), 67% A (59–62 min), 67–90% A (62–65 min). The HPLC chromatograms of RU, HY, ISQ, HI, MY, QOG and QU detected at wavelength of 254 nm and NAA, CHA and CAA detected at wavelength of 330 nm were shown in Fig. S1 and S2 (Supplementary data).

2.4

2.4 Total flavonoid and total phenolic contents

The total flavonoid contents of each sample were determined by Zou et al., 2004. 1 mL of sample was mixed with 0.3 mL of 5% NaNO2 for 6 min, then 0.3 mL of 10% Al(NO3)3 was added and allowed to stand for 6 min, after that 4 mL of 4% NaOH was added into the mixture. After 15 min incubation at room temperature, it was in a constant volume of 10 mL with 60% ethanol, and the reaction mixture absorbance was measured at 510 nm. Around 1 mL of 60% ethanol was used as a blank instead of 1 mL of sample. The total flavonoid contents were expressed as grams of rutin equivalents per 1 g of the extract (RE mg/g extract, y = 0.8726x – 0.065, R2 = 0.9995).

The total phenolic contents of each sample were determined using the Folin-Ciocalteu method (Wu et al., 2010). 40 μL of sample was mixed with 1800 μL of FC reagent to stand for 5 min at room temperature, then 1200 μL of 7.5% Na2CO3 was added to incubate for 90 min in the dark. After reaction, the total phenolic contents were measured at 765 nm and expressed as mg of gallic acid equivalents (mg GAE/g extract, y = 1.3709x + 0.0411, R2 = 0.9994). All the measurements were taken in triplicate.

2.5

2.5 Antioxidant activity

2.5.1

2.5.1 DPPH radical scavenging activity

DPPH radical scavenging activity of each examined sample was performed by the method proposed by Wu et al., 2009. A series of the extracts with different dilutions (0.1 mL in 60% ethanol) were mixed with 60% ethanol solution (1.4 mL) and 0.004% DPPH ethanol solution (1 mL). The mixed solution was incubated in the dark for 70 min and measured the absorbance value at 517 nm. Vc was used as the positive control. The DPPH radical scavenging activity was calculated using the equation: DPPH scavenging activity (%) = (ADPPH – Asample/ADPPH) × 100.

2.5.2

2.5.2 ABTS radical scavenging activity

ABTS radical scavenging activity was used to evaluate the total antioxidant capacity. Different concentrations of Trolox (0.15–1.5 mM) or extract (10 μL) was added into ABTS working solution (200 μL), which was produced by combining the ABTS stock solution with 2.45 mM potassium persulfate in the dark at room temperature for 12–16 h and diluted with 80% ethanol to reach the absorbance of 0.70 ± 0.05 at 734 nm. The mixture was incubated for 2–6 min to determine the absorbance at 734 nm in the dark. The results were expressed as mmol/g Trolox equivalent antioxidant capacity (TEAC).

2.5.3

2.5.3 Reducing power

The measurement of reducing power was executed as the method by Wu et al., 2010 with some modifications. Different dilutions of extracts or BHT (0.5 mL) was blended with 0.2 M phosphate buffer (pH 6.6, 0.5 mL) and 1 % K3[Fe(CN)6] (0.5 mL). The mixed solution was incubated at 50 ℃ for 20 min, then 10% trichloroacetic acid (TCA) was added and centrifuged at 3000 rpm for 10 min. Then, distilled water (0.5 mL) and 0.1% FeCl3 (0.1 mL) were successively added into the supernatant (0.5 mL), shaken well, reacted at room temperature for 10 min, and the absorbance was measured at 700 nm.

2.5.4

2.5.4 FRAP

The FRAP method was implemented according to the work instruction of Beyotime FRAP assay kit. Different concentrations of Trolox (0.15–1.5 mM) or extract (5 μL) was incubated with 180 μL of FRAP working solution for 3–5 min at 37 °C in the dark, and then measured the absorbance at 593 nm. The results were expressed as mmol FeSO4 equivalent per gram of dry weight.

2.6

2.6 Cytotoxicity assay

2.6.1

2.6.1 Cell preparation and culturing

RAW264.7 macrophage cells, purchased from the CBCAS (Cell Bank of the Chinese Academic of Sciences, Shanghai, PR China), were cultured in DMEM (Invitrogen) containing 10% (v/v) fetal bovine serum (Hyclone) and antibiotics (100 U/mL penicillin and 100 μg/mL strepto-mycin) (Hyclone) at 37 °C in a humidified 5% CO2 incubator.

2.6.2

2.6.2 Cell viability assay

Cell viability was determined by MTT assay. Briefly, RAW264.7 cells were seeded into 96-well plates at a density of 1 × 104 cells per well 24 h before treatment. Cells were treated with various concentrations of HMLF crude extracts in the presence or absence of LPS (1 μg/mL) for 24 h followed by incubating with 5 mg/mL of MTT working solution for 4 h at 37 °C. Then added 100 μL of DMSO to dissolve the crystals, the absorbance of each well at 570 nm was measured. Three replicates were carried out for each of the different treatments.

2.6.3

2.6.3 NO level measurement

The nitric oxide (NO) content was measured by the Total Nitric Oxide Assay Kit (Beyotime, China) according to the manufacturer’s recommendations. RAW264.7 cells (5 × 105 cells/well) were plated into 24-well plates and incubated overnight. Then, cells were pretreated with extracts for 24 h and stimulated with lipopolysaccharide (LPS) at 0.5 μg/mL for 24 h. To measure NO secretion, the cell supernatant (50 μL) was harvested and reacted with the Griess reagent (50 μL) for 10 min at room temperature in the dark. The absorbance at 540 nm was detected and the NO level was calculated using a nitrite standard solution. The total amount of NO can be calculated by the total amount of nitrate and nitrite with the above method.

2.7

2.7 Statistical analysis

All results were expressed as mean values ± standard deviation (n = 3). Analysis of variance was performed by ANOVA procedure. p < 0.05 was considered as statistically significant. Pearson correlations between variables were established by SPSS (21.0). Besides, principal component analysis (PCA) was performed to analyze the interrelationships among the TFC values, TPC values, measured antioxidant activities, anti-inflammatory activity and bioactive compound contents, described the variation of harvest time using the same statistical software.

3

3 Results and discussion

3.1

3.1 Variations in total flavonoid and total phenolic contents at different harvest time

Phenolic compounds are ubiquitous in most medicinal herbs and are an important part of human diet due to their antioxidant and other health-promoting properties (Pandey and Rizvi, 2009; Balasundram et al., 2006). Therefore, the total flavonoid and phenolic contents of HMLF extracts at different harvest time were investigated as shown in Fig. 1 and Table 1.

Changes in the total flavonoid contents (A), total phenolic contents (B), ABTS radical scavenging activities (C) and FRAP (D) of HMLF at different harvest time.
Fig. 1
Changes in the total flavonoid contents (A), total phenolic contents (B), ABTS radical scavenging activities (C) and FRAP (D) of HMLF at different harvest time.
Table 1 Total flavonoids, total phenolics, DPPH, ABTS, reducing power and FRAP of HMLF at different harvest time.
Date Total flavonoids
(RE mg/g DW)		 Total phenolics
(GAE mg/g DW)		 DPPH IC50a
(mg/mL)		 ABTSb
(mmol/g Trolox)		 Reducing power
IC50a (mg/mL)		 FRAPc (mmol
FeSO4/g DW)
6.21 56.011 ± 1.176 23.180 ± 0.845 0.181 ± 0.005 1.399 ± 0.038 0.122 ± 0.005 3.327 ± 0.149
6.28 52.286 ± 1.829 18.303 ± 0.523 0.229 ± 0.009 1.042 ± 0.047 0.203 ± 0.005 2.975 ± 0.095
7.05 37.675 ± 1.130 20.036 ± 0.459 0.224 ± 0.008 1.175 ± 0.060 0.186 ± 0.006 3.062 ± 0.122
7.12 33.950 ± 1.425 12.760 ± 0.486 0.236 ± 0.006 0.987 ± 0.038 0.209 ± 0.009 2.824 ± 0.071
7.19 35.956 ± 1.348 18.689 ± 0.934 0.238 ± 0.011 0.934 ± 0.053 0.218 ± 0.007 2.752 ± 0.096
7.26 53.146 ± 1.701 19.023 ± 0.551 0.215 ± 0.005 1.309 ± 0.055 0.136 ± 0.006 3.149 ± 0.142
8.02 45.124 ± 1.645 30.290 ± 1.873 0.160 ± 0.003 1.570 ± 0.058 0.101 ± 0.003 3.644 ± 0.109
8.09 49.421 ± 1.680 25.195 ± 0.919 0.166 ± 0.005 1.416 ± 0.039 0.112 ± 0.004 3.377 ± 0.152
8.16 44.264 ± 1.784 16.225 ± 0.464 0.224 ± 0.009 1.165 ± 0.052 0.188 ± 0.005 3.049 ± 0.097
8.23 33.091 ± 0.893 15.577 ± 0.358 0.255 ± 0.009 0.844 ± 0.043 0.235 ± 0.008 2.308 ± 0.092
8.30 41.399 ± 1.615 10.797 ± 0.411 0.266 ± 0.007 0.753 ± 0.029 0.249 ± 0.011 2.045 ± 0.051
Vc – – 0.064 ± 0.003 1.034 ± 0.059 NDd 2.483 ± 0.087
BHT – – NDd NDd 0.136 ± 0.004 NDd

Each value is expressed as mean ± standard deviation (n = 3).

a IC50 value, the concentration at which the antioxidant activity inhibition ratio reached 50%;
b ABTS were expressed as mmol/g Trolox equivalent antioxidant capacity (TEAC);
c FRAP are expressed as mmol FeSO4 equivalents per gram of DW;
d ND, not determined.

Total flavonoid content (TFC) of HMLF extracts varied widely, ranging from 33.091 to 56.011 RE mg/g DW (Table 1). The results revealed that higher level of TFC was observed on 6.21 (June 21th, 56.011 RE mg/g DW) followed by 7.26 (July 26th, 53.146 RE mg/g DW) and 6.28 (June 28th, 52.286 RE mg/g DW). However, the HMLF extract on 8.23 (August 23th, 33.091 RE mg/g DW) registered much lower amounts of TFC. The TFC was arranged as following sequence: 6.21 > 7.26 > 6.28 > 8.09 > 8.02 > 8.16 > 8.30 > 7.05 > 7.19 > 7.12 > 8.23.

The total phenolic content (TPC) is related with the antioxidant activity of plant samples as described in many literatures (Zhang et al., 2017; Sapirstein et al., 2013). Hence, the TPC variation results of HMLF extract at different harvest time were analyzed and shown in Fig. 1B and Table 1. The TPC of HMLF extract examined in this study ranged from 10.797 to 30.290 GAE mg/g DW. The results revealed a considerable diversity in the TPC among the various extracts investigated in the present study. The highest TPC was found on 8.02 (August, 2th, 30.290 GAE mg/g DW), while the TPC on 8.30 (August 30th) was relatively low (10.797 GAE mg/g DW), which was much lower than other harvest times. Moreover, the TPC on 8.09 (August, 9th) and 6.21 (June, 21th) also reached 25.195 GAE mg/g DW and 23.180 GAE mg/g DW, respectively. The TPC of HMLF extract at each harvest time was in order: 8.02 > 8.09 > 6.21 > 7.05 > 7.26 > 7.19 > 6.28 > 8.16 > 8.23 > 7.12 > 8.30.

From the results of TFC and TPC, the extracts on 6.21 (June, 21th), 8.02 (August, 2th) and 8.09 (August, 9th) had higher TFC and TPC than other harvest time. The comparison showed that June was the primary stage of plant, and the temperature and precipitation were lower and less, which was not conductive to plant growth and metabolism. However, in August, due to the dual pressure of drought and temperature, large amounts of secondary metabolites in plants were accumulated, so as to remove the increased reactive oxygen in plant cells, which was conducive to the increase of TFC and TPC in plants. Therefore, it was appropriate to harvest the plant materials HMLF in early August.

3.2

3.2 Variations in antioxidant activity at different harvest time

3.2.1

3.2.1 DPPH radical scavenging activity

DPPH radical scavenging activity has been applied to measure the antioxidant activity of active ingredients in HMLF samples at different harvest time. The measurement results of the scavenging ability of the extracts towards the stable DPPH radical were presented in Table 1. The antioxidant activity of positive control Vc was favorable with IC50 value of 0.064 mg/mL, and the samples harvested on 8.02 (August, 2th), 8.09 (August, 9th) and 6.21 (June, 21th) possessed the highest DPPH radical scavenging activity, followed by 7.26 (July, 26th), 7.05 (July, 5th), 8.16 (August, 16th), 6.28 (June, 28th), 7.12 (July, 12th), 7.19 (July, 19th), 8.23 (August, 23th) and 8.30 (August, 30th). The results obtained were similar to those of TPC, which indicated that polyphenols in plants were closely related to their antioxidant activity. Besides, as shown in Table 1, the free radical scavenging capacity of DPPH was expressed as IC50 value, which is negatively correlated with antioxidant activity. Among the eleven samples, the IC50 value of 8.02 (August, 2th, 0.160 mg/mL) was the lowest, while the IC50 values of the remaining samples were not significantly different. It also showed that the sample on early August was effective in discoloring the radical solution raising to approximately 80% at relatively low concentrations. Meanwhile, the antioxidant activity of samples at other harvest time (7.26, 7.05, 8.16, 6.28) were similar to that of sample on early August, indicating that these samples harvested from July to August had excellent potentials for antioxidant activity.

3.2.2

3.2.2 ABTS radical scavenging activity

ABTS radical scavenging activity assay is the most common method used to evaluate the antioxidant activity of medicinal plants, and the results are expressed as the Trolox equivalent antioxidant capacity (TEAC). As demonstrated in Fig. 1C and Table 1, the ABTS radical scavenging activity of the samples harvested at different time throughout the flowering period from highest to lowest in the following order: 8.02 > 8.09 > 6.21 > 7.26 > 7.05 > 8.16 > 6.28 > 7.12 > 7.19 > 8.23 > 8.30, which was consistent with the results of DPPH free radical scavenging activity assay. The TEAC values for the eleven samples ranged from 0.753 to 1.570 mmol/g Trolox. Besides, the TEAC values of the samples harvested on 8.02, 8.09, 6.21, 7.26, 7.05, 8.16 and 6.28 were 1.570, 1.416, 1.399, 1.309, 1.175, 1.165 and 1.042 mmol/g Trolox, respectively, which were higher than the value of Vc (1.034 mmol/g Trolox). Therefore, the sample harvested on early August possessed strong ABTS radical scavenging activity.

3.2.3

3.2.3 Reducing power

According to literature, the reducing power of plant materials has a strong correlation with their antioxidant activity. In chemistry, reducing power represents the ability to donate electrons, which can effectively decrease oxidation intermediates, thus protecting organisms from oxidation pressure and playing an antioxidant role. The absorbance value at 700 nm increased with the increasement of the concentration of HMLF sample, which indicated that the reducing power was enhanced. It was illustrated that the reducing power of eleven samples and BHT. The sample IC50 values on 8.02 (August, 2th), 8.09 (August, 9th) and 6.21 (June, 21th) were 0.101, 0.112 and 0.122 mg/mL, which were significantly lower than positive control BHT (0.136 mg/mL), indicating these samples exhibited strong reducing power. HMLF extract is natural source, and the same family plant Abelmoschus manihot L. is a medicinal and edible plant, which can also be a source of tea and active ingredients. Hence, HMLF can be a natural source of antioxidant to substitute synthetic antioxidants in food and other industries.

3.2.4

3.2.4 FRAP

The principle of FRAP assay is that Fe2+ is reduced to Fe3+, and the total antioxidant capacity of the sample is represented by FeSO4 as the equivalent (Fig. 1D). As presented in Table 1, the FRAP activity of the samples ranged from 2.045 to 3.644 mmol FeSO4/g DW. The HMLF samples harvested on 8.02 (August, 2th), 8.09 (August, 9th) and 6.21 (June, 21th) had the highest FRAP activity, which were in agreement with the results of the above three antioxidant experiments. Simultaneously, almost all the samples possessed higher FRAP activity than positive control Vc (2.483 mmol FeSO4/g DW), except for 8.23 (August, 23th) and 8.30 (August, 30th) samples had slightly lower FRAP activity (2.309 mmol FeSO4/g DW and 2.045 mmol FeSO4/g DW). The results of FRAP activity showed that the HMLF samples had excellent antioxidant activity.

3.3

3.3 Variations in anti-inflammatory activity at different harvest time

The viability of macrophages is the basis of their immune function, which directly affects their function and response. MTT assay was applied to determine the cytotoxicity activity of HMLF crude extracts at different harvest time on RAW264.7 cells after treatment with LPS and various concentrations of extract. Fig. 2 showed that the concentrations of the crude extract at 50–300 μg/mL were chosen for the measurement of cell viability. It can be seen that there was no significant difference among the eleven crude extracts treated for 24 h, which indicated that the cytotoxicity activity was not significantly affected by up to 300 μM HMLF crude extract (>80% cell viability).

Cytotoxic effect of HMLF crude extract at different harvest time on the cell viability of RAW264.7 cells. The cells were treated with different concentrations of HMLF crude extract (50–300 μg/mL) and the viability was measured by MTT assay. The results are presented as mean ± S.D, and the experiments were repeated as triplicates.
Fig. 2
Cytotoxic effect of HMLF crude extract at different harvest time on the cell viability of RAW264.7 cells. The cells were treated with different concentrations of HMLF crude extract (50–300 μg/mL) and the viability was measured by MTT assay. The results are presented as mean ± S.D, and the experiments were repeated as triplicates.

Nitric oxide (NO) was important in the immune system as a kind of significant signal transduction medium, and was recognized as a mediator and regulator in the pathological reactions in inflammatory (Liu et al., 2017). The anti-inflammatory effects of HMLF crude extract on LPS-induced NO production in RAW264.7 cells were investigated using Griess assay. Fig. 3. showed that LPS group significantly enhanced NO production, which indicated that the inflammatory model was established successfully. The NO production level was inhibited obviously after treatment by HMLF crude extracts. In particular, treatment with extract harvested on 8.02 (August, 2th) significantly inhibited LPS-induced NO production from 8.659 μM to 3.569 μM with an inhibition ratio of 58.78%. Moreover, the extracts harvested on 7.26 (July, 26th) and 8.09 (August, 9th) also possessed good capacity to inhibit LPS-induced NO production of 4.780 μM and 4.488 μM. These results obtained indicated that the HMLF crude extract possessed anti-inflammatory activity, and could be applied as effective anti-inflammatory agent.

Effect of HMLF crude extract at different harvest time on the NO production in LPS-induced RAW264.7 cells. The results are presented as mean ± S.D, and the experiments were repeated as triplicates. LPS+, LPS-induced HMLF crude extract at different harvest time; 1, 6.21 (June, 21th); 2, 6.28 (June, 28th); 3, 7.05 (July, 5th); 4, 7.12 (July, 12th); 5, 7.19 (July, 19th); 6, 7.26 (July, 26th); 7, 8.02 (August, 2th); 8, 8.09 (August, 9th); 9, 8.16 (August, 16th); 10, 8.23 (August, 23th); 11, 8.30 (August, 30th). ###p < 0.001 compared with the control group. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the HMLF crude extract-treated group.
Fig. 3
Effect of HMLF crude extract at different harvest time on the NO production in LPS-induced RAW264.7 cells. The results are presented as mean ± S.D, and the experiments were repeated as triplicates. LPS+, LPS-induced HMLF crude extract at different harvest time; 1, 6.21 (June, 21th); 2, 6.28 (June, 28th); 3, 7.05 (July, 5th); 4, 7.12 (July, 12th); 5, 7.19 (July, 19th); 6, 7.26 (July, 26th); 7, 8.02 (August, 2th); 8, 8.09 (August, 9th); 9, 8.16 (August, 16th); 10, 8.23 (August, 23th); 11, 8.30 (August, 30th). ###p < 0.001 compared with the control group. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the HMLF crude extract-treated group.

3.4

3.4 Variations in organic acids and flavonoids contents at different harvest time

Phenolic compounds of HMLF harvested at eleven different time were conducted to quantitative analysis by HPLC, and Fig. 4 showed the variations of ten main compounds contents (organic acids and flavonoids). As can be seen from Fig. 4, the contents of NA, HY, ISQ, HI, MY, QOG and QU were more affluent than those of CHA, CAA and RU, which were detected at lower quantities. The highest contents of NA and HY appeared on 6.21 (June, 21th) were 0.509 and 9.431 mg/g, respectively. The relatively rich ingredients ISQ and QOG harvested on 8.02 (August, 2th) ranged 4.709–13.977 mg/g and 0.919–3.834 mg/g. High HI content was also detected on 8.09 (August, 9th) at 10.761 mg/g. Nevertheless, MY and QU reached their highest contents (0.672 and 1.400 mg/g) on 7.05 (July, 5th). In addition, relatively low contents of CHA peaked at 0.495 mg/g on 7.26 (July, 26th), while CAA and RU peaked at 0.233 and 0.438 mg/g on 8.02 (August, 2th). On the whole, the harvest period with high content of active components in HMLF was early August (38.676 mg/g of the total amount of ten compounds), which possessed the high level of HY, ISQ, HI and QOG that can facilitate the antioxidant and anti-inflammatory activities of HMLF sample. In conclusion, early August may be the optimal harvest time window according to the results obtained in section 3.1–3.4.

The contents of ten active compounds in HMLF samples at different harvest time. a The content of HY, ISQ, HI, QOG and QU; b The content of NA, CHA, CAA, RU and MY.
Fig. 4
The contents of ten active compounds in HMLF samples at different harvest time. a The content of HY, ISQ, HI, QOG and QU; b The content of NA, CHA, CAA, RU and MY.

Due to the relatively dry and cold in the region where the samples collected in this experiment, the monthly average temperature and precipitation in Harbin from May to September are shown in Fig. S3. Generally, spring has lower temperature and less rainfall. In summer, the temperature is high and humid, while the temperature drops rapidly in autumn. The accumulation of secondary metabolites in plants is affected by environmental factors, including temperature, precipitation, sunshine and other factors. Heat-map of active compounds contents in HMLF samples from different harvest time was generated to elaborate the variation of secondary metabolites during the growth process of HML (Fig. S4). The temperature and precipitation in May and June were lower, which was not conductive to the growth and metabolism of the plants. In July, the temperature and precipitation increased significantly, which made the plants grow and metabolize rapidly, thus increasing the secondary metabolites to a certain extent. However, in August, due to the dual stress of temperature and drought, large amounts of secondary metabolites were accumulated in plants. Therefore, the contents of bioactive components in August were relatively high (Fig. 4). In addition, water shortage can change the oxidation balance in plants to adapt to drought conditions. Thus, a certain amounts of reactive oxygen species (ROS) were generated in plants, which had a positive effect on the synthesis of secondary metabolites.

Growth-differentiation equilibrium hypothesis holds that under the condition of abundant environmental resources, plants are dominated by growth, while under the condition of scarce resources, plants are mainly differentiated. This is because the growth and ontogeny of plants can be divided into two processes of growth and differentiation at the cellular level. The secondary metabolites of plants are the products of the physiological process of cell maturation and specialization. Therefore, under appropriate environmental conditions, plants give priority to differentiation, synthesis and accumulation of a large number of secondary metabolites (Peter et al., 2001). This is consistent with the hypothesis of resource acquisition, plants will grow slowly and generate high secondary metabolites under harsh natural conditions. On the contrary, plants will grow faster and produce low secondary metabolites under favorable conditions, which is the result of natural selection (Byers, 2000). When it comes to the autumn of September, due to the decrease of temperature and precipitation, it is more unfavorable to the growth of plants, and at the same time, the plants enter the fruiting period, which is not conducive to the harvesting of flowers. Therefore, August is suitable for harvesting HMLF with high content of bioactive ingredients.

3.5

3.5 Principal component analysis

Principal component analysis (PCA) is a statistical analysis process for identifying the data correlations. PCA was performed to further analyze the relationships among TPC values, TFC values, antioxidant activities, anti-inflammatory activity and bioactive compound contents. These first two principal components represented the direction at which the data showed the largest and second largest variations that explained 78.65 % of the total variability (Fig. 5). PC 1 and PC 2 were estimated from the eigenvectors of the correlation matrix represented 63.01% and 15.64%, respectively. PC 1 = 0.475 TFC + 0.972 TPC – 0.940 DPPH + 0.909 ABTS – 0.877 RP + 0.899 FRAP – 0.858 AIFT + 0.693NA + 0.168 CHA + 0.838 CAA + 0.812 RU + 0.875 HY + 0.905 ISQ + 0.740 HI + 0.731 MY + 0.857 QOG + 0.495 QU PC 2 = –0.461 TFC – 0.130 TPC + 0.221 DPPH – 0.331 ABTS + 0.397 RP – 0.283 FRAP + 0.440 AIFT – 0.069NA – 0.770 CHA + 0.422 CAA + 0.294 RU + 0.250 HY + 0.133 ISQ + 0.564 HI + 0.477 MY + 0.024 QOG + 0.593 QU

Principal component analysis loading plot of TFC, TPC, antioxidant activity and active compounds from different harvest time HMLF samples (A), Principal component analysis score plot of different harvest time HMLF samples (B), Principal component analysis Biplot of different harvest time HMLF samples (C). RP, reducing power; AIFT, anti-inflammatory activity; TFC, total flavonoids content; TPC, total phenolics content; NA, neochlorogenic acid; CHA, chlorogenic acid; CAA, caffeic acid; RU, rutin; HY, hyperin; ISQ, isoquercetin; HI, hibifolin; MY, myricetin; QOG, quercetin–3′–O–glucoside; QU, quercetin.
Fig. 5
Principal component analysis loading plot of TFC, TPC, antioxidant activity and active compounds from different harvest time HMLF samples (A), Principal component analysis score plot of different harvest time HMLF samples (B), Principal component analysis Biplot of different harvest time HMLF samples (C). RP, reducing power; AIFT, anti-inflammatory activity; TFC, total flavonoids content; TPC, total phenolics content; NA, neochlorogenic acid; CHA, chlorogenic acid; CAA, caffeic acid; RU, rutin; HY, hyperin; ISQ, isoquercetin; HI, hibifolin; MY, myricetin; QOG, quercetin–3′–O–glucoside; QU, quercetin.

The PC 1 correlated with TPC, DPPH, ABTS, reducing power, FRAP, contents of neochlorogenic acid, caffeic acid, rutin, hyperin, isoquercetin, hibifolin, myricetin, quercetin–3′–O–glucoside with loadings of 0.972, –0.940, 0.909, –0.877, 0.899, –0.858, 0.693, 0.838, 0.812, 0.875, 0.905, 0.740, 0.731 and 0.857, respectively. The PC 2 related to contents of chlorogenic acid and quercetin with loadings of –0.770 and 0.593 (Fig. 5A). In addition, it can also be found that DPPH, ABTS, reducing power, FRAP activity, anti-inflammatory activity and TPC values were highly correlated with coefficient correlations of –0.923, 0.891, –0.875, 0.861 and –0.900, respectively, which indicated that PC 1 was closely related to antioxidant and anti-inflammatory activities (Table S1). Chlorogenic acid and quercetin had higher loadings on PC 2, but the antioxidant activity of PC 2 was not high, which indicated that chlorogenic acid and quercetin in HMLF samples had little contribution to antioxidant activity. This phenomenon was that although chlorogenic acid and quercetin have good antioxidant activity, the contents of chlorogenic acid and quercetin in HMLF samples were relatively low, so the variations of chlorogenic acid and quercetin had little effect on the antioxidant activity of HMLF samples.

PCA can also provide an overview of the qualities of eleven HMLF samples at different harvest time. The samples harvested on 7.05 (July, 5th) and 7.26 (July, 26th) were highly correlated with PC 2, which was favorable to collect the samples with higher contents of chlorogenic acid and quercetin (Fig. 5B). Additionally, sample at the right part of Fig. 5B was harvested on 8.02 (August, 2th), which positively correlated with PC 1. This sample are best described with high antioxidant activities, anti-inflammatory activity and high contents of bioactive compounds. Therefore, early August was more suitable for harvesting HMLF samples than other time.

4

4 Conclusions

This article firstly reported the phytochemical analysis, antioxidant and anti-inflammatory activities of Hibiscus manihot L. flower extracts at different harvest time. The results of HPLC analysis indicated that hyperin, isoquercetin, hibifolin and quercetin–3′–O–glucoside were the most abundant flavonoids in HMLF. Moreover, DPPH radical scavenging activity (IC50 0.160 mg/mL), ABTS radical scavenging activity (1.570 mmol/g Trolox), reducing power (IC50 0.101 mg/mL) and FRAP (3.644 mmol FeSO4/g) test results of HMLF sample harvested on August, 2th possessed stronger antioxidant activities. The cytotoxicity of the early August sample against RAW264.7 cells in vitro testified it was safe to RAW264.7 cells at the concentrations of 50–300 μg/mL. Anti-inflammatory activity results showed that the extracts harvested on August, 2th exhibit a significant effect on NO production (3.569 μM) in LPS induced RAW264.7 cells, which was slightly higher than the control group (2.073 μM), indicating that the HMLF crude extract had certain anti-inflammatory activity and could be used as an effective anti-inflammatory agent. The PCA also indicated that the quality of early August HMLF samples was better than the other samples. Therefore, Hibiscus manihot L. flower could be recognized as a bioactive natural product which deserves to be further investigated for its mechanism of action in biological activities evaluation and other chemical ingredients. This study revealed that the Hibiscus manihot L. flower harvested on early August contained high content of organic acids and flavonoids, and exhibited strong antioxidant capacity and anti-inflammatory activity, which can be applied to develop new functional or healthy ingredients.

CRediT authorship contribution statement

Ju-Zhao Liu: Formal analysis, Visualization, Writing – original draft, Writing – review & editing. Chun-Chun Zhang: Investigation. Yu-Jie Fu: Conceptualization, Writing – review & editing. Qi Cui: Conceptualization, Methodology, Data curation, Writing – original draft, Visualization, Writing – review & editing.

Acknowledgements

The authors gratefully acknowledge the financial supports by National Key R & D Program of China (2017YFD0600706, 2016YFD0600805), Fundamental Research Funds for the Central Universities (2572018AA17, 2572016AA48, 2572019EA01, 2572018CT01, 2572017EA03), China Postdoctoral Science Foundation (2021M692893), Traditional Chinese Medicine Modernization Special Project of Zhejiang Traditional Chinese Medicine Science and Technology Plan (2021ZX008) and Dr. Philippe Golfier from Heidelberg University for proofreading. We appreciate the great experimental support from the Public Platform of Medical Research Center, Academy of Chinese Medical Science, Zhejiang Chinese Medical University.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Appendix A

Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2021.103503.

Appendix A

Supplementary data

The following are the Supplementary data to this article:

Supplementary data 1

Supplementary data 1


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