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Article

Bioactivity-Guided Screening of Wound-Healing Active Constituents from American Cockroach (Periplaneta americana)

by
Juan-Juan Zhu
1,
Shun Yao
2,
**n Guo
1,
Bi-Song Yue
1,
**u-Ying Ma
3 and
**g Li
1,*
1
Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610065, China
2
Department of Pharmaceutical and Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu 610065, China
3
Sichuan Gooddoctor-Panxi Pharmaceutical Co., Ltd., **chang 615000, China
*
Author to whom correspondence should be addressed.
Molecules 2018, 23(1), 101; https://doi.org/10.3390/molecules23010101
Submission received: 12 December 2017 / Revised: 30 December 2017 / Accepted: 31 December 2017 / Published: 20 January 2018
(This article belongs to the Collection Bioactive Compounds)
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Abstract

:
Ethanol extract (EE) from Periplaneta americana (PA) is the main ingredient of Kangfuxin, which is a popular traditional chinese medicine (TCM) and has long been used for the clinical treatment of burns, wounds and ulcers. We compared the wound-healing activities of three extracts of PA using cutaneous wound-healing in mice as the bioactivity model. These three extracts were EE, total polysaccharide and total protein. We also tracked bioactive fractions in the EE by organic reagent extraction, column chromatography and HPLC. Seven compounds were successfully identified from the water elution fraction of the EE of PA using UPLC-MS. Among these compounds, four compounds (P2, P3, P4, P5(1)) were first reported in PA. Some of these compounds have been previously reported to have various pharmacological activities that could contribute to the high wound-healing activity of PA.

1. Introduction

Wound healing is a complex biological process including three classic stages: inflammation, new tissue formation and remodeling [1]. Any aberrancies at each stage can lead to delayed wound healing. Many synthetic drugs and artificial skins are used for wound healing, but they are expensive and readily cause allergic reactions in sensitive people [2,3,4]. Thus, there is a real need for an alternative to synthetic wound-healing products. Natural products are the most reliable and successful sources of drug leads. For example, insects have been increasingly used as effective bioactive products and as they are one of the most diverse taxa of living organisms can provide a considerable resource of potential alternatives [5]. Therefore, the potential for insect constituents, such as cockroaches, to enhance natural wound healing is a particularly important research avenue.
Periplaneta americana (PA), the American cockroach, has the largest body size in the family Blattidae. It is also one of the most famous sanitation-related insects with strong vitality and successful reproduction [6]. PA is widely distributed in subtropical and tropical regions across the world [7]. Many previous PA studies have focused on the infestation of human dwellings and the strong ability to transmit pathogenic fungi [8]. However, other physiological and pharmacological studies have demonstrated that PA constituents have favorable tissue-repairing [9], antibacterial [10], antitumor [11] and immunity-enhancing activities [12]. Additionally, this insect has been used in traditional chinese medicine (TCM) as an important biomedical component for the treatment of TCM syndromes such as blood stasis, ulcers, burns and wounds for hundreds of years [13]. Consequently, the formulations of many TCM preparations, such as Kangfuxin Liquid, Ganlong Capsule, ** slowly after 2 h of modeling, respectively, while **gwanhong ointment was evenly applied onto the wound area with medical cotton stickers of the PC group mice. The wounds were left open after administering either the treatment or control, and the mice were put back into clean cages. The administration for each group was conducted once a day for 9 days. In the process of modeling and administration, the mice all showed a good mental state and activity.

3.5.2. Evaluation of Wound-Healing Activity and Statistical Analysis

The cutaneous wounds of mice were photographed, and the wound areas were measured by tracing the margins after the injury (day 0), on the third, sixth and ninth days. The percentage of wound healing was calculated with the following formula [41]:
Percentage of wound healing (%) = (original area − unhealed area)/original area × 100
The wound-healing percentage was analyzed by one-way ANOVA followed by Tukey’s HSD post hoc test using statistical software package (SPSS) and GraphPad Prism software (version 5.0, GraphPad Software Inc., San Diego, CA, USA). All results are displayed as mean ± standard deviation (SD), and p < 0.05 was considered as statistically significant.

4. Conclusions

Through the combination of traditional separation strategies with bioactivity tracking, we successfully found that the water elution fraction (Fr.D1) of EE of PA had the highest healing activity. Seven compounds (including cyclopeptide, diterpenoid, phenolic acid, fatty acids and glycosides) were identified from this fraction with the UPLC-MS method, among which the diterpenoid, one phenolic acid and the two glycosides were first reported in PA. Most of the identified compounds have been reported to have antibacterium, anti-inflammation or enhancing immunity activities that were highly likely to be associated with wound repair for mice. The study is expected to lay the foundation for further research and utilization of PA.

Supplementary Materials

The supplementary materials are available online.

Acknowledgments

This work was supported by the Sichuan Gooddoctor-Panxi Pharmaceutical Company Limited and Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University.

Author Contributions

Juan-Juan Zhu, Shun Yao and **g Li designed the experiments; Juan-Juan Zhu performed the experiments; Juan-Juan Zhu and **n Guo analyzed the data; **u-Ying Ma provided experimental materials; Juan-Juan Zhu wrote the paper. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Sample Availability: Samples of the used insect materials are available from the authors.
Molecules 23 00101 g001 550
Figure 1. Wound-healing assay of mice treated with ethanol extract (EE), total polysaccharide (TPS), total protein (TP), **gwanhong (positive control—PC) and pure water (negative control—NC) at 0, 3, 6 and 9 days. (a) Photographs of dorsal excisional skin wounds on different days. Day 0 pictures were taken immediately after wounding; (b) Data are given as mean ± standard deviation (SD) for three mice in each group. Statistical analysis of wound area used one-way ANOVA followed by Tukey’s Honestly Significant Difference (HSD) post hoc test. The characters indicate statistically significant differences (p < 0.05): a, b and c: EE compared with other four groups on days 3, 6 and 9; d: between TP/TPS and NC on day 3.
Figure 1. Wound-healing assay of mice treated with ethanol extract (EE), total polysaccharide (TPS), total protein (TP), **gwanhong (positive control—PC) and pure water (negative control—NC) at 0, 3, 6 and 9 days. (a) Photographs of dorsal excisional skin wounds on different days. Day 0 pictures were taken immediately after wounding; (b) Data are given as mean ± standard deviation (SD) for three mice in each group. Statistical analysis of wound area used one-way ANOVA followed by Tukey’s Honestly Significant Difference (HSD) post hoc test. The characters indicate statistically significant differences (p < 0.05): a, b and c: EE compared with other four groups on days 3, 6 and 9; d: between TP/TPS and NC on day 3.
Molecules 23 00101 g001
Molecules 23 00101 g002 550
Figure 2. UPLC chromatogram (five peaks) (a) and total ion chromatogram (seven peaks) (b) at 254 nm (here are shown only intercept chromatograms for the first 7 min because there was no peak after then).
Figure 2. UPLC chromatogram (five peaks) (a) and total ion chromatogram (seven peaks) (b) at 254 nm (here are shown only intercept chromatograms for the first 7 min because there was no peak after then).
Molecules 23 00101 g002
Molecules 23 00101 g003 550
Figure 3. Separation and analysis procedure of ethanol extract from Periplaneta americana (PA).
Figure 3. Separation and analysis procedure of ethanol extract from Periplaneta americana (PA).
Molecules 23 00101 g003
Table
Table 1. The yield, color and healing rate of different solvent extracts (Fr.A: chloroform fraction; Fr.B: ethyl acetate fraction; Fr.C: n-butanol fraction; Fr.D: water fraction D) of 500 mL of Periplaneta Americana (PA) ethanol extract.
Table 1. The yield, color and healing rate of different solvent extracts (Fr.A: chloroform fraction; Fr.B: ethyl acetate fraction; Fr.C: n-butanol fraction; Fr.D: water fraction D) of 500 mL of Periplaneta Americana (PA) ethanol extract.
ExtractYield (%) *Color of ConcentrateHealing Rate (Mean ± SD)% **
Fr.A0.063Red brown9.4 ± 1.4
Fr.B0.104Red brown75.4 ± 3.7
Fr.C0.864Tan61.5 ± 5.6
Fr.D11.111Black brown93.6 ± 4.7
* The yield rate means the proportion of various solvent extracts in the PA powder. ** The healing rate of various extracts on day 9 (pure water—negative control group: 71.3 ± 3.4%; **gwanhong—positive control group: 92.3 ± 5.8%). SD: standard deviation.
Table
Table 2. Comparison of water fraction Fr.D and water elution fraction Fr.D1 on day 9 in wound-healing (PC: positive control group; NC: negative control group) activities.
Table 2. Comparison of water fraction Fr.D and water elution fraction Fr.D1 on day 9 in wound-healing (PC: positive control group; NC: negative control group) activities.
FractionColor *Healing Rate (Mean ± SD)% *
Fr.DRed92.3 ± 2.8
Fr.D1Light pink99.5 ± 0.8
PCLight pink92.6 ± 2.3
NCReddish-brown71.3 ± 3.4
* The color and healing rate of wounds on day 9. SD: standard deviation.
Table
Table 3. Compounds identified from the water elution fraction (Fr.D1) of Periplaneta Americana (M.F.: molecular formula).
Table 3. Compounds identified from the water elution fraction (Fr.D1) of Periplaneta Americana (M.F.: molecular formula).
No.Ion Peakm/zCompoundM.F.StructureReference
P1[M + K]+235.1451Cyclo-(l-Val-l-Pro)C10H16N2O2 Molecules 23 00101 i001[20]
P2[M + H]+337.04582-(4′-Methyl-3′-pentene)-6-hydroxymethyl-10-methyl-12-hydroxyl-(2,6,10)-triendodecanic acidC20H32O4 Molecules 23 00101 i002[21]
P3[M + H]+273.0745ArbutinC12H16O7 Molecules 23 00101 i003[22]
P4[M + H]+259.07854-Benzyloxy-3-methoxybenzoic acidC15H14O4 Molecules 23 00101 i004[23]
P5(1)[M + H]+263.1840(E)-3-Hexenyl-β-d-glucopyranosideC12H22O6 Molecules 23 00101 i005[24]
P6[M + K]+339.05397-Hydroxycotadeca-noic acidC18H36O3 Molecules 23 00101 i006[25]
P7[M + Na]+331.1576(S)-2,3-Dihydroxypropyl hexadecanoic acid esterC19H38O4 Molecules 23 00101 i007[7]

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MDPI and ACS Style

Zhu, J.-J.; Yao, S.; Guo, X.; Yue, B.-S.; Ma, X.-Y.; Li, J. Bioactivity-Guided Screening of Wound-Healing Active Constituents from American Cockroach (Periplaneta americana). Molecules 2018, 23, 101. https://doi.org/10.3390/molecules23010101

AMA Style

Zhu J-J, Yao S, Guo X, Yue B-S, Ma X-Y, Li J. Bioactivity-Guided Screening of Wound-Healing Active Constituents from American Cockroach (Periplaneta americana). Molecules. 2018; 23(1):101. https://doi.org/10.3390/molecules23010101

Chicago/Turabian Style

Zhu, Juan-Juan, Shun Yao, **n Guo, Bi-Song Yue, **u-Ying Ma, and **g Li. 2018. "Bioactivity-Guided Screening of Wound-Healing Active Constituents from American Cockroach (Periplaneta americana)" Molecules 23, no. 1: 101. https://doi.org/10.3390/molecules23010101

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