Discovery of Anti-inflammatory Ingredients in Chinese Herbal Formula Kouyanqing Granule based on Relevance Analysis between Chemical Characters and Biological Effects

Abstract

Kouyanqing Granule (KYQG) is a traditional Chinese herbal formula composed of Flos lonicerae (FL), Radix scrophulariae (RS), Radix ophiopogonis (RO), Radix asparagi (RA), and Radix et rhizoma glycyrrhizae (RG). In contrast with the typical method of separating and then biologicalily testing the components individually, this study was designed to establish an approach in order to define the core bioactive ingredients of the anti-inflammatory effects of KYQG based on the relevance analysis between chemical characters and biological effects. Eleven KYQG samples with different ingredients were prepared by changing the ratios of the 5 herbs. Thirty-eight ingredients in KYQG were identified using Ultra-fast liquid chromatography-Diode array detector-Quadrupole-Time-of-flight-Tandem mass spectrometry (UFLC-DAD-Q-TOF-MS/MS) technology. Human oral keratinocytes (HOK) were cultured for 24 hours with 5% of Cigarette smoke extract (CSE) to induce inflammation stress. Interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumour necrosis factor-α (TNF-α) were evaluated after treatment with the eleven KYQG samples. Grey relational analysis(GRA), Pearson's correlations (PCC), and partial least-squares (PLS) were utilized to evaluate the contribution of each ingredient. The results indicated that KYQG significantly reduced interleukin-1β, interleukin-6, interleukin-8, and tumour necrosis factor-α levels, in which lysine, γ-aminobutyric acid, chelidonic acid, tyrosine, harpagide, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isoquercitrin, luteolin-7-o-glucoside, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, angoroside C, harpagoside, cinnamic acid, and ruscogenin play a vital role.

Figure 1. Chemical structural formula of the identified ingredients in KYQG (except for the conjectural saponin 1 and saponin 223).

Figure 2

UFLC-Q-TOF-MS/MS characters (A) and cluster analysis results (B) of eleven KYQG samples. Twenty-five ingredients were confirmed by the retention time. The MS data of the reference substances and thirteen ingredients were characterized by MS data and related studies. Given the rescaled distance of 5, eleven KYQG samples could be divided into eight classes as follows: S2 and S4 belonged to one class, S3 and S5 belonged to one class, S9 and S10 belonged to one class, and the remainder of the samples constituted their own class.

Figure 3. The effects of KYQG (the original formula) on IL-1β, TNF-α, IL-8, and IL-6 production.

Dex: Dexamethasone; NP: Niuhuangjiedu Pian; KYQG: Kouyanqing Granule. Groups: control group, model group, NP group (10 μg/mL), Dex groups (1 and 10 μM), and KYQG groups (5.6, 55.6, and 555.6 μg/mL). The control group and model group received the same volume of RPMI-1640 medium for the treatment. Each bar represents the contents of IL-1β, TNF-α, IL-8, and IL-6 as the mean ± SD, n = 3. ^*P < 0.05 and ^**P < 0.01 vs. control group, ^#P < 0.05 and ^##P < 0.01 vs. model group.

Figure 4. The effects of eleven KYQG samples on IL-1β, TNF-α, IL-8, and IL-6 production.

Dex: Dexamethasone; NP: Niuhuangjiedu Pian; KYQG: Kouyanqing Granule. Groups: control group, model group, NP group (10 μg/mL), Dex group (1 μM), and eleven KYQG samples groups (55.6 μg/mL). Control group and model group received the same volume of RPMI-1640 medium for the treatment. Each bar represents the contents of IL-1β, TNF-α, IL-8, and IL-6 as the mean ± SD, n = 3. ^*P < 0.05 and ^**P < 0.01 vs. control group, ^#P < 0.05 and ^##P < 0.01 vs. model group.